Let’s rock…
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Brännvall, M., Kikovska, E., Wu, S. & Kirsebom, L.A. (2007). Evidence for induced fit in RNase P RNA mediated cleavage, J. Mol. Biol. 372, 1149-1164.
II
Wu, S., Chen, Y., Lindell, M., Guan Zhong, M. & Kirsebom, L.A.
(2011). Functional coupling between a distal interaction and the cleavage site in bacterail Rnae P RNA mediated cleavage. J. Mol. Biol.411, 384-396
III
*Sinapah, S., *Wu, S., *Chen, Y., B. M. F. Pettersson, V. Gopalan &
Kirsebom, L.A. (2010). Cleavage of model substrates by archaeal
RNase P: role of protein cofactors in cleavage-site selection. Nucl. Acids Res. 39, 1105-1116.
IV
Wu, S., Kikovska, E., Lindell, M. & Kirsebom, L.A. (2011). Cleavage
mediated by the catalytic domain of Rnase P RNA. Manuscript.
V
Wu, S., Chen, Y., Guanzhong Mao, Trobro, S., Kwiatkowski, M. &
Kirsebom, L.A. (2011). The -1 residue and transition-state stabilization in RNase P RNA-mediated cleavage. Manuscript
* Shared first authorship
Reprints were made with permission from the respective publishers.
Contents
Introduction.....................................................................................................9
Divalent Metal Ions / Mg2+.......................................................................11
Processing and Maturation of tRNA ........................................................14
RNase P ....................................................................................................16
RNase P and the RNA subunit.............................................................16
Different types of RNase P and the conserved parts within them .......17
Substrates of RNase P..........................................................................18
Divalent metal ions and RNase P ........................................................19
Interactions between RNase P RNA and its substrate .........................21
Different model substrates used in my study ...........................................25
A glance at the kinetics ............................................................................26
About this thesis .......................................................................................30
Induced fit model ..........................................................................................31
The two domains of the RNase P RNA ........................................................37
Contribution of the S-domain...................................................................37
Contribution of the C-domain ..................................................................40
What is happening around the cleavage site? ...............................................43
The contribution of the -1 residue in the substrate ...................................44
Concluding remarks ......................................................................................49
Swedish Summary ........................................................................................51
Acknowledgements.......................................................................................54
References.....................................................................................................56
Abbreviations
A
C
C5
DNA
G
RNA
RNase P
RPR
RPP
M1RNA
PFU
pre-tRNA
tRNA
U
adenine
cytosine
Escherichia coli RNase P subunit
deoxyribonucleic acid
guanine
ribonucleic acid
ribonuclease P
RNase P RNA
protein subunit of the RNase P
Escherichia coli RNase P RNA
Pyrococcus furiosus RNase P
precursor tRNA
transfer RNA
uracil
Introduction
As is well known, DNA (Deoxyribonucleic acid) is the molecule that carries
the genetic information in almost all organisms’ cells (with the exception of
RNA virus). The inherited genetic information is then transcribed to another
molecule in cell, RNA (Ribonucleic acid). By intricate processes, RNA is
then translated into a more complex molecule, protein. This process from
DNA to RNA to protein is called the central dogma of molecular biology
(Figure 1).
Transcription
DNA
ReverseTranscription
Translation
RNA
Protein
Figure 1. Central dogma of molecular biology. The genetic information carrier DNA
is transcribed to RNA: RNA can also be reverse-transcribed back to DNA. RNA is
then translated to protein.
Proteins are very important molecules in organisms. They do the most of the
work, for example in the human cell. Proteins can be hormones, enzymes,
cell signals and they can establish the structure of our body and more. Nonetheless all the information that encodes protein is carried by DNA. DNA
carries the biological information that is inherited from parental cells and
DNA that is not for the genetic coding can have structural purpose or be
involved in regulating gene expression. DNA is a more stable molecule
compare to RNA because it has –H instead of –OH at position 2’ on the sugar ring and it usually has two complementary strands. However, from studies
in recent years RNA actually appears to be a very important molecule in its
own right and not a disposable copy of the DNA.
In fact, RNA is considered a more ancient biological molecule compare to
DNA and protein. There is much evidence supporting this hypothesis, which
is called “the RNA world” (Gesteland et al. The RNA World, Third edition).
No matter what happened in the past, RNA is still serving paramount roles in
cellular processing as we discovered in recent years. The discovery of Ribozymes (see below), Riboswitches (Bastet et al. 2011) and RNA interfer-
9
ence (RNAi) (Ketting 2011; Perrimon 2010), are among the important discoveries that have opened up the era of the RNA world.
Table 1. List of known naturally existing ribozymes.
Large
ribozymes
Name of the ribozyme
Found presenting
species
Biological function
Divalent Metal
ion depended
Peptidyl transferase
23S rRNA
All species
Decoding mRNA into
protein
Yes
RNase P
Almost all species
tRNA processing
Yes
Group I introns
In bacteria, higher
plants, phage and
lower eukaryotes
Intron splicing
Yes
Intron splicing
Yes
Twin-ribozyme intron
organization
Yes
Lead toxicity
Yes
Replication of the
satellite RNA
No
Satellite RNA and
Viroid RNA excision
No
Group II introns
GIR1 branching
ribozyme
Leadzyme
Hairpin ribozyme
Small
ribozymes
Hammerhead ribozyme
In plants, fungi,
bacteria, and lower
eukaryotes
In myxomycete
Didymium iridis
and several species
of the amoebaflagellate Naegleria
In 5S rRNA of
cellular mammalian ribosomal
In RNA satellites
of plant viruses
In plant viroids,
helminths, archaebacteria and
eubacteria
HDV ribozyme
In virus
Viroid RNA excision
Yes
Mammalian CPEB3
ribozyme
In mammals
mRNA processing
No
VS ribozyme
In mitochondrial
DNA of Neurospora
Satellite RNA excision
No
glmS ribozyme
In some Grampositive bacteria
Gene regulation/
riboswitch
No
CoTC ribozyme
Eukaryotic
Transcription termination of RNA polymerase II
Yes
Ribozyme (ribonucleic acid enzyme) is a key finding that opened the RNA
world hypothesis that was first named by Kelly Kruger and colleagues in
1982 (Kruge et al. 1982). Ribozyme is a kind of enzyme. Unlike protein
enzyme, the catalytic subunit of ribozyme is made of RNA. Actually the
ribosome, where the protein is translated from the mRNA code, has RNA as
the core active site (Nissen et al. 2000). Furthermore it has been shown that a
10
highly deproteinized ribosome is still active (Noller et al. 1992). Thus the
ribosome is indeed a ribozyme. In nature ribozymes can either catalyze the
hydrolysis reactions on their own phosphodiester bonds or on phosphodiester
bonds of another RNA, with or without help of the protein subunit. Some of
the ribozymes can also catalyze the reverse reaction, for example VS ribozyme (Lilley, 2011). In vitro selected ribozymes have also been reported
to be able to catalyze peptide bond formation (Tamura 2011; Zhang et al.
1997).
Already known naturally existing ribozymes are listed in table 1.
Divalent Metal Ions / Mg2+
Metal ions play a very important role in the catalysis of the ribozymes. For a
better understanding of ribozyme catalysis, it is essential to talk about metal
ions, especially divalent metal ions in big ribozymes.
Divalent metal ions are essential for enzyme activity in the cell. In the cell
one of the most abundant divalent metal ion is Mg2+. It exists in all cell types
and all organisms. The concentration of Mg2+ in animal cells is 30 mmol/L
(Ebel et al. 1980). In plant, the concentration is higher. Over 300 enzymes
need the presence of Mg2+ for their catalytic activity. These enzymes appear
in all metabolic pathways. For example, ATP and T7 RNA polymerase are
all Mg2+-dependent (Yang et al. 1980). In general, Mg2+ is involved in catalytic activity by interacting with the substrate or the enzyme, or both. Mg2+
interacts with the substrate or the enzyme through inner or outer sphere coordination, by altering the conformation of the enzyme, by stabilizing anions
or reactive intermediates, by binding to ATP thereby activating the molecule
to act as nucleophile and eventually making the chemical reaction occur.
Manganese (Mn2+) is a divalent metal ion that can replace Mg2+ in some
conditions, for example in enzymes catalysis. Chemically Mn2+ is similar to
Mg2+. In our experiment (see below) we also used Mn2+ to replace Mg2+ and
it gave interesting results.
Divalent metal ions are also important in catalysis of ribozymes. As table 1
shows, most of the small ribozymes do not need divalent metal ions for their
catalytic activity. Data suggest that monovalent cations are enough for the
catalysis of these small ribozymes. The cleavage mechanism of ribozymes
can be called general acid-base catalysis and it involves SN2 reaction. In
cleavage mechanism of small ribozymes adjacent internal nucleotides outside the cleavage site act as proton donors and acceptors. The cleavage products become a 2',3'-cyclic phosphate and a 5'-hydroxyl group (Wilson et al.
2011; Murray et al. 1998).
11
In contrast, not like some small ribozymes that can only rely on monovalent
ions, divalent metal ions are important for the bigger ribozymes, like Group I
and Group II introns and RNase P, to have efficient catalysis (Adams et al.
2004; Toor et al. 2008). Therefore these ribozymes are called metalloenzymes. However monovalent ions are disposable for catalysis of bigger ribozymes. For example in RNase P-mediated catalysis, monovalents are
needed. Monovalents can also help to shield the negatively charged RNA
backbone and facilitate in RNA folding. However, the preference for monovalent ions and their exact mechanism are still unclear.
Divalent metal ions play important roles in ribozyme catalysis in two ways:
1) Folding and stabilizing the structure.
Correct folding is crucial for enzymes catalytic activity. The reason why
proteins can fold into rigid three-dimensional structure in water-based solution is because they have hydrophobic residues. Nucleic acids do not have
the hydrophobic residues to help them fold. Instead nucleic acids have a
negatively charged phosphate backbone. This makes the folding of nucleic
acid more difficult because of the repulsion effects caused by this backbone.
Divalent metal ions can neutralize the negatively charged backbone to some
extent and thereby help the folding.
Data also show that divalent metal ions can stabilize the structure of the
RNA by coordinating to 2’OHs and bases, thus stabilizing the wobbly base
pairs and the backbone (Shi et al. 2000).
2) Stabilizing transition state conformations, thereby generating the nucleophile and its involvement in the chemistry of the cleavage.
Efficient and accurate catalysis not only depends on the structure of the molecule in a steady state, but also relies on the transition state conformations.
Cleavage of RNA is possible because nucleophile attacks the phosphodiester
backbone and hydrolyzes the chemical bond. Metal ions are involved in
cleavage in various ways: 1). A metal-coordinated hydroxide ion can act as a
general base or Lewis acid to abstract the proton or accelerate the deprotonation, respectively. 2). Stabilize the 3’-oxygen leaving group. 3). Help the
nucleophilic attack by either stimulating the phosphorus center or stabilizing
the charged trigonal-bipyramidal intermediate (Yasuomi et al. 2001). Different large ribozymes have different mechanisms for cleavage, although their
chemical mechanisms all involve SN2 displacement reaction. For large ribozymes Group I and II introns share a similar general acid-base catalysis
mechanism. They both have two-step splicing mechanism. In the first step,
in Group I intrions, the 3’-OH group of the attacking external G residue is
the nucleophile and attacks on the 5’ splice site. In Group II introns, the 5’
12
splice site is attacked by the 2’-OH of the internal A residue or by a hydroxide ion. The second step involves an attack from the 3’-OH of the 3’-end
upstream exon on the 3’ splice site to make the final splicing products. Metal
ions were suggested to serve roles in the reaction by their association with a
water molecule. Mg2+ in particular was suggested to coordinate to substrate
oxygens, then activate the nucleophile, and stabilize the scissile phosphate
and the charge on the leaving group. The two-metal-ion mechanism that was
seen in protein enzymes was suggested to be the mechanism of the Group I
and Group II intron splicing (Yasuomi et al. 2001; Piccirilli et al. 1992; Stahley et al. 2006; Forconi et al. 2009; Steitz and Steitz 1993). In this model,
two divalent metal ions that are 3.9Å apart were suggested to facilitate the
catalysis. Both divalent metal ions act as Lewis acids that can stabilize the
chemical transition state. One ion activates the attacking water or sugar hydroxyl. Another one is suggested to stabilize the oxyanion leaving group
(Steitz and Steitz 1993).
RNase P has also been suggested to use a two-metal-ion mechanism in the
cleavage reaction. The RNase P-substrate complex structure that was solved
recently also suggests that there are at least two distinct metal ions that play
an important role in the cleavage (Reiter et al. 2010). However, apart from
the roles of the divalent metal ions in the cleavage mechanism, RNase P in
general has a different mechanism in catalysis compared to Group I and II
intron. In RNase P-induced cleavage, the deprotonized water molecule acts
as the nucleophile. It was suggested that the 2’OH at the -1 residue acts as an
outer (or inner) sphere ligand for Mg2+ (Persson et al. 2003; Brännvall et al.
2004; Zahler et al. 2005). It is possible that divalent metal ion (Mg2+) coordinating 2’OH also uses the 3’ bridging oxygen and the pro-Rp-oxygen as
coordinator in RNase P RNA-mediated cleavage. Particularly pro-Rpoxygen has been observed in type A, type B and eukaryal RNase Ps (Chen
1997; Thomas et al. 2000) (for a description of the different types of RNase
P, see below). Divalent metal ions were also suggested to play roles in stabilizing and generating the nucleophile, stabilizing the transition states and
stabilizing the leaving group (Smith D. et al. 1993; Guerrier-Takada et al.
1986; Warnecke et al. 1996, 2000). The products after cleavage by RNase P
remain 5’-phosphates and 3’- hydroxyls. Note that divalent metal ions can
also induce hydrolysis of the RNA to yield products with 5’-hydroxyls and
2’;3’-cyclic phosphates. Thus in RNase P-mediated cleavage, RNase P has
to prevent the divalent metal ion-induced hydrolysis as a result of a nucleophilic attack from the 2’-OH side and to favor the cleavage from the other
side operated by a hydroxide ion coordinated by a divalent metal ion. However, the exact mechanism of RNase P-mediated cleavage remains unclear
(Fig. 2).
13
Regarding the divalent metal ions, we were interested in answering the following questions: 1) What is the role of the divalent metal ions (Mg2+) in
RNase P RNA catalysis? 2) How many functional Mg2+ ions are involved in
the catalysis and what are their roles? 3) What is the relevance of Mg2+ in the
structure/ formation changes of the RNase P RNA and substrates? In my
thesis I will discuss these questions.
H
Figure 2. A model of reaction catalyzed by RNase P RNA through two-metal-ion
mechanism. One divalent metal ion coordinated hydroxyl ion acts as the nuclophile.
Another divalent metal ion stabilize the oxyanion leaving group. Two metal ions
both act as Lewis acids and stabilize the pentacoordinated transition state. The products after cleavage are 3’OH and 5’phosphate.
Because divalent metal ions can induce cleavage on the backbone of RNA
and the binding sites of the divalent metal ions are specific, divalent metal
ions can be used as tools to map the metal-ion binding site on RNA. Pb2+ has
been used frequently in footprint experiments to investigate the structural
change of the RNA (Brännvall et al. 2001; Doctor Thesis of Magnus Lindell).
In our study we also used Pb2+-induced cleavage in footprint experiments to
explore the structural changes of the RNase P RNA or the substrates (see
below). For example, we used footprint experiments to explore the conformational change of the RNase P RNA after we displaced some nucleotides.
Processing and Maturation of tRNA
When the tRNA is transcribed in the cell, it has extra nucleotides on both the
5’ and 3’ ends of the pre-tRNA. These extra nucleotides need to be removed
by enzymes so that the pre-tRNA is matured and functional for translation
(Figure 4.).
14
There are several ribonucleases, depending on the origin, involved in the
processing at the 3’ end of a pre- tRNA (Schurer et al. 2001 and Mörl &
Marchfelder, 2001). tRNase Z is the main enzyme for maturation of the 3’end (Hartmann, et al. 2009). In bacteria, the endoribonucleases RNase E,
RNase G, and numbers of other exonucleases are responsible for tRNA 3’end processing (Li et al. 1996; 2002). Moreover, for these tRNAs without
the encoded 3’-CCA motif or where the 3’-CCA motif is damaged after 3’
maturation, tRNA nucleotidyl transferase adds the 3’-CCA motif to the
tRNA (Wen et al. 2005).
However, at the 5’-end, RNase P is the only enzyme that is responsible for
the maturation of a pre-tRNA. RNase P can cleave on the substrates irrespective of the existence of the 3’end (Tous et al. 2001). RNase P in most of
these cases cleaves the substrate between the -1 and +1 residues, except for
tRNAHis and tRNASeCys ; these are cleaved between -2 and -1 residues (Orellana et al. 1986; Burkard et al. 1988a, 1988b; Green. et al. 1988; Kirsebom et
al. 1992). After cleavage by RNase P, the cleavage products become a 3’ –
OH and a 5’-phosphate, respectively (as mentioned before). This applies to
both presence and absence protein part of the RNase P.
The
objectives of my thesis work have been to investigate substrate
interaction and mechanism of cleavage by the trans acting ribozyme RNase P RNA. First, I will give a general overview of the field and
relevant factors that influence cleavage efficiency and choice of cleavage site.
This will be followed by a brief summary of the strategy and methods that I
have used in my studies. Finally, I will discuss the specific projects during
my thesis work.
15
RNase P
RNase P and the RNA subunit
There are about 1000 copies of RNase P RNA (also is called P RNA or
RPR) in the fast growing E. coli cell and are responsible for tRNA precursor
processing in all three kingdoms of life (Dong et al. 1996). In eukaryotes,
RNase P is located in the nucleus. The kinetics of RNase P-mediated catalysis includes substrate binding, chemical cleavage, and product release, followed by new rounds of substrate binding. Thus RNase P is a turn-over ribozyme. Some of the ribozymes, like the Hammerhead ribozyme, generate
cleavage on itself. However, RNase P, like the ribosome and other protein
enzymes, has another RNA as its substrate and acts in trans. Therefore
RNase P is a true enzyme.
Bacterial RNase P has two subunits: one RNA subunit and one protein subunit. The number of the protein subunits varies, depending on the origin of
the organism. For example, there are four to five protein subunits in the Archaea and at least nine in the Eukarya (Kirsebom. 2007; Kirsebom et al.
2009). It is worth mentioning that in human mitochondria no RNA part of
the RNase P has been found so far and protein alone has the RNase P property (Kufel et al. 2008; Holzmann et al. 2008). This might be very important
in terms of evolution.
Early, people thought the RNA part of the RNase P was just a scaffold for
the enzymatically active protein to attach to and get the right configuration.
After an experiment by Cecilia Guerrier-Takada, it was clear that the RNA
part of the RNase P had catalytic activity (Guerrier-Takada et al. 1983). In
fact, the RNA part of the RNase P is the catalytic subunit of the enzyme. In
vitro, most RNase P RNA can perform enzymatic activity without the help
of the protein part (Thomas et al., 2000; Kikovska. et al. 2007), but in vivo
the protein part is indispensable.
It is already known that in bacteria the protein part of RNase P binds on the
5’ leader of the precursor to stabilize the RNase P-substrate complex to increase the affinity of the substrate and catalytically important metal-ion
binding (Crary et al. 1998; Sun et al. 2006). In bacterial RNase P, the protein
part is also involved in product release, prevention of product rebinding,
cleavage site recognition, broadening the substrate selection and stabilizing
the native structure of RNase P RNA (Tallsjö and Kirsebom 1993; Liu et al.
1994; Park et al. 2000; Buck et al. 2005). However, the recently published
co-crystal structure indicates that the protein is near the 5’ end of tRNA, but
not in direct contact with it. Instead, the protein sits between P15 and P3
stem of RNase P RNA and also contacts the CR-IV and CR-V regions (Rei16
ter et al. 2010). There is little known about the function of archaeal and eukaryal RNase P proteins. Interestingly, the eukaryotic RNase P holoenzyme
cleaves on the tRNAHis precursor and leaves 7-bp-long acceptor stem (for a
review see Kirsebom and Torobro 2009). This is in contrast to the bacterial
RNase P RNA and the human RNase P RNA where the RNase P RNAs generated cleavage leaves an 8-bp-long acceptor stem (Orellana et al. 1986,
Kikovska et al. 2007).
RNase P is essential for life and exists in all kinds of organisms. It is a universally conserved ribozyme. This implies that RNase P is perhaps a remnant of the RNA world and has been important in evolution. However there
are still a few exceptional species where no RNase P has been identified. For
example, there are no candidates for the genes that code for the RNase P
RNA or the protein in Aquifex aeolicus, although evidence shows that some
RNA is involved in the RNase P-like performance in this organism (Kazakov et al. 1991; Lombo et al. 2008; Marszalkowski et al. 2008). Likewise, in
the archaeon Nanoarchaeum equitans, there is no RNase P found (Lennart et
al. 2008). There are also some other organisms that do not have RNase P,
like viruses or phage, but these organisms cannot grow on their own.
Different types of RNase P and the conserved parts within them
a
c
b
Type A
Type B
e
d
Type C
Type M
Figure 3. Secondary structure of different types of RNase P RNA from Bacteria and
Archaea, except for type P. a) E. coli RNase P RNA. b) Pyrococcus furiosus (Pfu)
RNase P RNA. c) Bacillus subtils RNase P RNA. d) Thermomicrobium roseum
RNase P RNA. e) Methanocaldococcus jannaschii RNase P RNA. The type of
RNase P RNA that each of them belongs to is stated underneath. The picture is
based on the figure from RNase P database:
http://www.mbio.ncsu.edu/rnasep/home.html
RNase P is an ancient RNA molecule and is considered to be a relic of the
RNA world. During evolution Bacteria and Archaea RNase P RNA have
17
evolved into five different types: Type A, Type B, Type C, Type M and
Type P. RNase P in Eukaryotes has a more complicated situation and has not
been divided to classes.
A type means ancestral type. E. coli RNase P RNA, i.e. M1 RNA belongs to
this type. Most of the bacteria and archaea RNase P RNAs also belong to
this type.
B type was named because it was first discovered in Bacillus. It exists in
most Firmicutes.
C type means the Chloroflexi type.
M type means the Methanococci type. This type only belongs to Archaea
RNase P RNA. The RNA part of this type RNase P is not proficient in catalysis in vitro without the protein part.
P type is named from Pyrobaculum. This type has only been seen in the
species Pyrobaculum. It has almost no specific domain (S-domain) as in
other Bacteria and Archaea RNase P RNAs and it has less conserved parts
than other types of RNase P RNA (Lai et al. 2010). (Figure 3)
Although the sequences of the RNase P RNA are very different, a core of the
sequence and part of the secondary structure are still conserved within all the
RNAs. The P8 and L15 are involved in direct substrate binding, and the P4
and surrounding joining regions are the catalytic important regions (Figure
5). These regions are evolutionary conserved in A, B and M types of RNase
P RNA, and even in eukaryotic RNase P RNA (Kirsebom 2007).
All the RNAs (except for P type) have two domains: specificity domain (Sdomain) and catalytic domain (C-domain) (Figure 5). The crystal structures
for both type A and type B are available and the structures show that the Sdomain of these two types of RNase P RNA can fold into a different tertiary
structure (Kirsebom 2007; Haas and Browa, 1998; Kazantsev et al. 2005).
Substrates of RNase P
RNase P has many substrates other than precursor-tRNA in nature. These
substrates either have a single stranded RNA attached on the 5’end of a long
hairpin construct, like 4.5 S RNA (Bothwell et al. 1976), or they have a
tRNA-like construct, like tmRNA (Komine et al. 1999) or plant viral RNAs
(Guerrier-Takada et al. 1988). Moreover, it is also involved in the maturation
of some other noncoding RNAs such like 2S RNA, snoRNAs. In the leader
sequence of mRNA it is suggested that RNase P can recognize and cleave
riboswitchs such as the coenzyme B12 riboswitch in E. coli and Bacillus subtilis (Hori et al. 2000; Tous et al. 2001; Altman et al. 2005; Seif and Altman
2008). Among all these substrates tRNA precursors are the most abundant.
18
It was suspected that there had to be more substrates for RNase P in the cell.
The way to look for these substrates was to look for the accumulated precursors in the cells under the conditions when no RNase P is made (Yang and
Altman 2007; Samanta et al. 2006; Coughlin et al. 2008). In this fashion
some other potential substrates were found. For example, in some RNAs in
E. coli there are cleavage sites between ORF regions for RNase P. These
sites were suggested to be important for the degradation or regulation of the
operons. Nevertheless, it is not easy to recognize the substrates in nature due
to the conformational variation of the RNA in solution (Greenleaf et al. 2008)
or due to the transitional conformational changes it undergoes.
Many artificial model substrates have been used in RNase P research as well.
According to data the minimal size of the substrate in vitro that can be recognized and cleaved by RNase P should contain one extra nucleotide upstream of the canonical cleavage site, together with a 7-basepair stem and an
RCCA motif at the 3’end of the substrate (Werner M et al. 1997; Forster and
Altman 1990; Liu and Altman 1996). Although the RCCA motif is not absolutely required for cleavage by RNase P RNA in vitro, evidence shows that it
is important for the rate of cleavage (Svärd and Kirsebom, 1992). The substrate with two nucleotides upstream of the cleavage site has significantly
increased affinity to RNase P RNA and a faster cleavage rate compare to the
substrate that has only one nucleotide before the canonical cleavage site
(Crary et al. 1998). Nevertheless in paper I we report a small RNA, that has
only a 3- base pair stem, a 9-nucleotide 5’-leader and a 4- nucleotide loop,
which can act as substrate for the RNase P RNA in vitro (Detailed discussions below, Figure 7). In addition, there were reports demonstrating that the
bacterial RNase P holoenzyme cleaves small, single-strand RNA as well
(Loria et al. 2001).
Divalent metal ions and RNase P
As mentioned before, divalent metal ions, particularly Mg2+, are essential in
RNase P mediated catalysis, both in vivo and in vitro. There are about 100
Mg2+ ions bound to RNase P RNA (Beebe et al. 1996). Some of these Mg2+
ions contribute to RNase P RNA folding or they assist the interaction of the
RNase P RNA with its substrate, the interaction of RNA with protein, or
they are involved in the chemistry of cleavage. Note that the structural Mg2+
binding site is not necessary important for RNase P catalysis.
The functionality of RNA is very much dependent upon its correct folding, it
is the case for RNase P RNA and its substrate. Bacterial RNase P RNA is
catalytically active without the protein part, so the folding of its RNA part of
bacterial RNase P is independent of the protein part of the RNase P as well,
19
making the role of Mg2+ even more important. The folding of B. subtilis
RNase P RNA in vitro occurs at Mg2+ ion concentrations <3.2mM and the
optimal catalytic activity is at >50mM. M1 RNA folds at Mg2+ ion concentrations <2.4mM and the optimal catalytic activity is at >25mM (ProteinRwviews, V.10: Ribonuclease P, Fenyong Liu and Sidney Altman).
The role of the divalent metal ion is twofold in RNase P RNA-mediated
catalysis. Firstly, the divalent metal ions promote folding of the RNA to an
active conformation and secondly, they can promote chemical cleavage.
Other than Mg2+, Ca2+, Mn2+, Sr2+ and even Pb2+ can also induce folding and
promote cleavage of RNase P RNA to some extent. It is noteworthy that Pb2+
induced folding leads to a different structure than other divalent metal ions.
Pb2+ and Sr2+ cannot promote RNase P RNA catalysis at physiological pH by
themselves, but together they can (Brännvall et al. 2001). In nature, both
Mg2+ and Ca2+ are present in the cell, and possibly they can coordinate together to regulate the RNA-based activity as a function of their intracellular
concentration. For instance, Mg2+ can promote a faster catalysis than Ca2+in
RNase P-induced cleavage thereby regulating the biological processes in
which RNase P is involved.
Several important Mg2+ ions have been identified in RNase P RNA. The
Mg2+ on the P4 helix does not induce any conformational change of RNase P
RNA, but it is believed to contribute to a distal effect in the vicinity of the
cleavage site (see above). Divalent metal ions are also important in the TSL(T-stem-loop region) /TBS (T-stem- loop binding site) interaction (about
different interactions, see below). It has been claimed that the interaction of
TSL-/TBS changes the conformational position of the divalent metal ion in
the vicinity of the cleavage site (Brännvall et al. 2007). The RCCA-/ RNase
P RNA interaction also has divalent metal ion involved in. There are three
divalent metal ions in the P15-loop (Kazakov and Altman 1991; Kufel and
Kirsebom 1996; Zito K et al. 1993; Glenmarec et al. 1996). These metal ions
are suggested to stabilize the interaction formation of RCCA-/ RNase P
RNA as well as influence the divalent metal in the vicinity of the cleavage
site (Brännvall et al. 2004). A divalent metal ion is suggested to stabilize the
interaction of +73/294, and play a role in the surrounding of the exocyclic
amine of G+1 and 2’OH, N7 of base +73 of the substrate. There are divalent
metal ion(s) also in the vicinity of the cleavage site that is suggested to influence the conformation of A248/ N-1 interaction (Brännvall et al. 2007).
Additionally, it was suggested that the true substrate of RNase P should contain a divalent metal ion in the vicinity of the +1/+72 pair (Perreault and
Altman 1993).
20
Because of the importance of Mg2+ in RNase P RNA catalysis, determining
the Mg2+ profile and the optimal Mg2+ concentration is necessary for finding
out the characteristic of each RNase P RNA/substrate combination. Note that
cleavage rate increases as the Mg2+ concentration increases before reach a
plateau, therefore measuring the rate before the optimal Mg2+ concentration
(plateau) makes the data not comparable with each other. In paper II, we
used the optimized Mg2+concentration in the cleavage rate determination to
make the data more comparable than in paper I (see below).
Interactions between RNase P RNA and its substrate
acceptor-stem &
T-stem
RCCA motif
Cleavege site of
RNase P
D/ T loop
RNase P
Cleavage site
RCCA motif
Anticondon
D-stem &
anticodon-stem
& variable loop
Figure 4. Secondary and tertiary structure of matured tRNAphe. The secondary structure elucidates the different parts of the tRNA. The tertiary structure elucidates the
“L-shape” of the tRNAphe and the corresponding parts of the secondary structure.
(The secondary structure is modified from Wikipedia provided picture:
http://en.wikipedia.org/wiki/Transfer_RNA )
It has been suggested that the tRNA domain of the tRNA precursor has already folded to its characteristic structure at the precursor stage. RNase P
can recognize this structure and interacts with it (Chang 1973; Swerdlow et
al. 1984; Kirsebom 2007)
There are many interactions between RNase P RNA and the pre-tRNA.
These interactions are distributed on the acceptor stem and the T-arm of the
pre-tRNA (Kirsebom 2007). The matured tRNA is folded into an L-shape
tertiary structure (Kim et al. 1973) and the interactions with RNase P RNA
are all located on “upper arm” of the L-shape (see Figure 4).
21
S-domain
C-domain
Figure 5. The secondary structure of E. coli’s RNase P RNA. The region with the
dark circle indicates the conserved regions within different types of RNase P RNA.
The dotted line in the middle of the structure is the separation line of the S-domain
and the C-domain. The picture is modified from RNase P database,
http://www.mbio.ncsu.edu/rnasep/home.html.
These interactions are determinants which RNase P recognizes and binds to
result in accurate and efficient cleavage. RNAs that do not have the tRNA
like structures e.g., 4.5 S RNA, can still be recognized by RNase P even if
they do not have all the determinants (Altman & Kirsebom 1999). The more
determinants that the RNase P can interact with, the more accuratly and efficiently the RNase P can cleave its substrate (Svärd et al 1993).
22
The TSL-/TBS interaction
T-stem-loop region (TSL-region) is located at the “elbow” of the L-shape
tRNA (also referred to as the D/T loop domain). The part of RNase P RNA
that interacts with this domain is called TBS (T-stem- loop binding site).
This TBS binding site is within the conserved part of the specific domain
(P7-P11) of RNase P RNA (Krasilnikov et al. 2004; Pan et al. 1996; and
below) (see Figure 5). In B. subtilis, it has been suggested that the interaction with TSL is established through tertiary interaction of the 2’-OHs of
four residues (residue 54, 56, 61 and 62) and possibly a 4-amino group of
residue C57 (Loria et al. 1997) (see Figure 4). In the recently published
RNase P-tRNA co-crystal structure, these interactions were confirmed; in
particular, residues 56 and 19 in the TSL region interact with TBS region in
RNase P RNA (Reiter et al. 2010). In B. subtilis the residue corresponding to
A233 in E. coli RNase P RNA interacts with the 2’-OH at residue 64 in the
T-stem in the pre-tRNAphe (Pan et al 1995). Residue 118 in the specific domain is also part of the TBS region (Grandeur et al.1994). Moreover, A129,
A130, A180, G229 and G230 (E. coli RNase P RNA numbers) were found
to interact with the TSL region from the recently published crystal structure
(Reiter et al. 2010). The TSL/TBS interaction was believed to be a major
determinant for the specific binding of pre-tRNA (Loria et al. 1997) and the
crystal structure has shown that this is indeed the case (Reiter et al. 2010).
The RCCA–/RNase P RNA motif interaction
The RCCA is a preserved sequence in all species tRNA genes and it is located at the 3’end of tRNA. It is suggested to interact with the P15 loop of
RNase P RNA (Kirsebom and Svärd 1994; Okabe 2003). The +74C and
+75C pair with G293 and G292 in M1 RNA, respectively. These pairings are
believed to be Watson-Crick base paring, which is one of the way ribozymes
interact with their substrates. The other way of RNA-RNA association in
ribozyme catalysis is through tertiary structure interaction and recognition,
like TSL/ TBS interaction. Moreover it has also been suggested that U294 in
M1 RNA interacts with +73 in the tRNA precursor (Kikovska et al. 2005;
Brännvall et al. 2002, 2003, 2004). (see Figure 6) In the crystal structure
published in 2010, the authors suggested that U294 and G+73 also form a
base pair and the terminal A76 forms a weak interaction with G291 (Reiter
et al. 2010). The RCCA-/RNase P RNA interaction is proposed to anchor the
RNase P RNA on pre-tRNA, exposing the cleavage site and consequently
making the cleavage occur on the correct site.
Other interactions have been suggested in the vicinity of the RCCA interaction. A258 in the P-15 loop of M1 RNA, together with C75/G292 forms a
triple base pair (Heide et al. 1999, 2001). Also in addition, A76 is suggested
23
to interact with G259 through an interaction with the Hoogsteen edge (Heide.
2001).
M1 RNA
294
+73
Canonical
cleavage
site
tRNA
M1 RNA
Figure 6. A diagram of the interactions in the vicinity of cleavage site (the big arrow indicats the cleavage site). The RCC motif in tRNA base pairs with (U)GG in
M1 RNA. C+74C+75 pair with G293G292 through WC-base pairing, but the U294/G+73 is
not WC-base pairing. The -1U interacts with A248 from M1 RNA. The circled A, B
and C show the position of functionally important divalent metal ions. The figure is
modified from paper I.
The A248/ N-1 interaction
The majority of tRNA precursors in E. coli have U at the -1 position (the
residue right before the cleavage site). The A248 residue is suggested to
interact with the -1 residue in pre-tRNA (Zahler et al. 2003) (see Figure 6).
Data indicate that the interaction is perhaps not Watson-Crick base pairing.
The two oxygens at position 2 and 4 on the base are suggested to play an
important role (paper V), but in my study (paper IV and paper V), we found
that A248 perhaps is not interacting with N-1 (for more discussion see below). However, cross-linking data indicate that several residues are positioned close to the -1 residue. They are: A248, A249, C252, C253, G332 and
A333. These residues might form a binding pocket for the -1 residue in the
tRNA precursor (for review see Kirsebom, 2007). This interaction most likely works together with the RCCA/RNase P RNA interaction to help the
RNase P bind correctly and thereby facilitate the efficiently cleavage.
U69/+5 interaction and 2’OH/RNase P RNA interaction
The P4 helix (see Figure 5) has been suggested to be important for divalent
metal ion (Mg2+) binding (Hardt et al. 1995; Harris et al. 1995; Christian
2000; Kaye 2002). These Mg2+ ions are important for the function of RNase
P RNA. It is also suggested that residues in the vicinity of U69 from M1
RNA interact with the residue positioned 5 residues downstream of the cleavage site in the tRNA (Christian et al. 2006). This interaction can affect the
metal ion binding on the P4 and subsequently affects on the metal ion that is
on the cleavage site.
24
The 2’OH at the cleavage site is important for ground state binding, cleavage
rate, cleavage site recognition, and metal ion binding in the vicinity. Toria et
al. state that the 2’OH at the cleavage site mediates an interaction between
RNase P RNA and its substrate (Toria et al. 1998). Others believe that it is
the 2’OH that actually interacts with RNase P RNA (Brännvall et al. 2005,
2004; Zahler et al. 2005; Persson et al. 2003).
For a schematic diagram of the different interactions, I refer to Paper I.
Different model substrates used in my study
pTS-L (U-1)
Figure 7. Sketch of the secondary structure of different model substrates. The left
substrate represents the full length tRNA precursor. The upper right substrate is the
substrate that maintains T-stem/loop structure of the tRNAser precursor (the dotted
line on the pTS-L substrate indicates the cutting site from where the pATSer substrates are generated ). pATSerUGGAAA is derived from pATSerUG substrate with an
altered tetra T-loop. The lower right substrate does not have the T stem/loop structure. The figure is modified from paper I.
In my study, I used different substrates to increase our understanding of the
roles of various determinants in RNase P RNA-mediated catalysis. As described previously, RNase P RNA interacts with the tRNA precursor on the
acceptor stem and D/T stem loop. Hence we used the pATSer series of substrates which have the acceptor stem and T-loop, and the T-stem from pretRNA to form a hairpin-like structure (McClain et al. 1987). This pATSer
substrate therefore has the potential to interact with the TBS region. Our data
suggested that the remainder T-loop of the model substrate does indeed interact with the TBS region in M1 RNA (see discussion below and Paper I).
Because the TSL/TBS interaction is very important for RNase P RNA catalysis, we decided to generate another series of substrates. These were derived from the pATSer substrate but had 4 nucleotides to replace the T-loop
while maintaining the 12 nucleotides long stem from pATSer. This substrate
25
is called pATSerUGGAAA or pATSerCGGAAA, depending on the distinct residues at the -1 and +73 positions. For example: UG equals to -1U/+73G, and
CG is equivalent to -1C/+73G, etc. To be able to investigate the RNase P
RNA-substrate interaction further, we further minimized the stem in the
pATSerGAAA substrates to a 3-nucleotide pairing stem; this series of substrates is called pMini3bp substrate. (Figure 7)
All these model substrates, i.e. pATSer, pATSerGAAA, and pMini3bp, can act
as substrates of RNase P in vitro under both the physiological pH condition
and a pH condition where the chemistry of cleavage is suggested to be rate
limiting (Loria et al. 1998, Kikovska et al. 2005a, also see below).
A glance at the kinetics
In my study all the kinetic constants were measured at pH 6.1, the pH at
which the chemistry of cleavage is suggested to be the rate-limiting step. All
the rate constants, except for some data in paper I, were determined under
single turnover condition. This is a better condition than multiple turnover
for measuring RNase P RNA mediated catalysis because the productreleasing step is the rate limiting step (See below).
Multiple turnover reaction
kcat
E+S
k+1
k-1
ES
k+2
k-2
EP
k+3
k-3
E+P
kobs
Figure 8. Scheme of a 3-step enzymatic reaction. E = free enzyme. S = substrate. P
= product. ES = enzyme-substrate complex. EP = enzyme-product complex. k = rate
constant.
The enzymatic kinetics can be measured in two different states: steady-state
and transient state. The steady state of enzyme reaction means that the rate
of enzyme-substrate (ES) formation equals to the rate of ES breakdown. The
Michaelis-Menten equations are based on this state.
When the amount of substrate (S) is more than that of the enzyme (E), each
E has to make many “turnovers” to be able to cleave the substrates. This is
26
called multiple turnover. In this condition Km, the Michaelis constant, can be
written as shown here [Eq. 1].
k
Km
k2
1
(E
k1
0
ES ) S
ES
(1)
Km is the concentration of substrate when the speed of reaction reaches half
the maximum speed. If we draw a figure with [S] on the X-axis and rate on
the Y-axis, it is not difficult to see that the smaller the Km, the stronger the
interaction between enzyme and substrate. If the k2 is negligible, i.e., when
k2 << k-1, then the Km can be written as shown in [Eq. 2]. In this situation,
the Km can be considered as the dissociation constant of ES.
k
k
Km
1
(2)
1
kcat is the rate constant representing the speed of converting the ES to product (P). As Figure 8 indicates, kcat can be written like [Eq. 3 and Eq. 4].
1
k cat
k cat
1
k 2
Vmax
E
1
k3
1
k2
1
k3
(3)
(4)
kcat/Km is called the specific constant and it is an indicator of how well an
enzyme catalyzes different substrates. When k2>>k1, then kcat/Km=k+1.
Single turnover reaction
In an enzymatic reaction, the speed of the reaction is determined by the ratelimiting step. For example, if the product-releasing step is the rate limiting
step, the entire rate of the reaction is dependent on this step, no matter how
fast the other steps are. The limitation of studying steady-state kinetics in
RNase P-mediated catalysis is that the limiting step under steady-state at
saturated substrate concentration is tRNA product release, not pre-tRNA
cleavage (Reich et al. 1988; Tallsjö et al. 1993). Therefore, studying the
transient-state kinetics in RNase P RNA catalysis has a great advantage. This
can also be called single turnover kinetics, which means the concentration of
enzyme is much greater than that of the substrate. A single molecule of the
enzyme can cleave at most one substrate molecule. Note that the protein part
of the RNase P helps the product release, so the product releasing step might
not be the rate-limiting step for the holo-enzyme. However the kinetics of
the RNase P holoenzyme is still unclear.
27
At pH 6.1, the chemistry of cleavage (k2) is suggested to be the rate limiting
step in the RNase P RNA mediated reaction (Loria et al. 1997; Warnecke et
al. 1996). This has been inferred from pH titration experiments. We use single turnover conditions at pH 6.1 in our reaction system for the analysis of
RNase P RNA kinetics.
Compared to multiple turnover condition, kobs/Ksto in single turnover conditions is equal to kcat/Km. kobs reflects the rate of cleavage of the scissile bond,
which means kobs= k+2. According to the description above, kcat/Km =k+1,
therefore kobs/Ksto = k+1. At pH 6.1, single turnover RNase P RNA mediated
cleavage, we argue that if k-1>>k2, then Ksto Kd. (paper II, III and V).
To investigate if the experiment is occurring at k-1>>k2 condition, a pulsechase experiment (also called dilution experiment) was needed (see paper
III). The key idea of this experiment was to dilute the already initiated reaction into a vast amount of reaction buffer (about 200 times in our experiment) to see if the reaction was “pulsed” due to the dilution. In our experiment we saw that without dilution the cleavage product increased with time.
With dilution it did not (supplementary data in paper II, III and V). Thus, for
RNase P RNA mediated cleavage at pH 6.1, single turnover condition, Ksto
Kd.
Ground state binding and dissociation constant
Kd is the dissociation constant. appKd stands for apparent Kd, which is also
called ground-state binding constant (Kikovska et al. 2005)). This constant
means that when the binding is strong between the two subunits, the appKd is
small, and when the binding is weaker the appKd becomes bigger. For instance, the reaction for enzyme (E) binding the substrate (S) is carried as
E•S E+S; the equation for this reaction is [Eq. 5]:
Kd
E
S
ES
(5)
Hill coefficient
Hill coefficient originally is used to quantify the effect of a ligand binding to
a molecule. It expresses the degree of cooperativity of a ligand binding to an
enzyme or molecule.
In our research, we used the Hill coefficient to characterize the cooperativity
of Mg2+ in RNase P RNA mediated cleavage. The number of Hill coefficient
reflexes the number of functional important Mg2+ in the cleavage. We calcu28
lated the Hill coefficient from Mg2+ titration experiment. The Hill coefficient
is defined by [Eq. 6]
v Vmaxa h /(k h
ah )
(6)
After rearrangement it can be written [Eq. 7]
log v /(Vmax
v)
h log a log k h
(7)
In equation 6 and 7, v is the velocity under certain Mg2+ concentration. Vmax
is the maximum velocity detected in a set of Mg2+ titration experiment, h is
the Hill coefficient and a is the Mg2+ concentration that the v corresponds to.
29
About this thesis
RNase P is essential in the cell and therefore understanding the mechanism
of RNase P-mediated catalysis is important. It will help us to understand
cleavage by ribozymes in general, the evolution of RNA and it will help us
design new medical tools to cure, for example, diseases caused by different
bacteria.
Previous decades of studies have progressed our knowledge of RNase P
structure, RNase P RNA folding, RNase P-substrate recognition and binding,
the roles of divalent metal ions in RNase P RNA-mediated cleavage, the
kinetics and mechanism of RNase P catalysis, and RNase P in the perspective of evolution. However, there are still many unanswered questions that
needs to be investigated.
What is the basic chemistry of RNase P-induced cleavage? Which residues
or chemical groups in the vicinity of the cleavage site contribute to the cleavage? RNase P interacts with its substrate, but what is the interaction in the
intermediate stage and how much do these interactions contribute to the catalysis? What is the conformation of the RNase P RNA-substrate in the transition state and how does that conformation affect the cleavage? What is the
exact role of divalent metal ions? What is the contribution of the S-domain
and the C-domain of RNase P and what are their roles in an evolutionary
perspective? What is the role of the proteins of the RNase P?
In my study we used biochemical tools to answer some of these questions.
Different constructs and mutants of RNase P RNA and model substrates
were used in my studies to provide information. We compared data from
Mg2+ profiles, cleavage site selection and cleavage rate of different RNase P
RNA-substrate combination. From these data we rationalize the possible
mechanisms in RNase P catalysis.
In this thesis I will mainly focus on the RNA part of the RNase P. My work
has contributed to the understanding of the RNase P RNA-substrate interaction, the function of the two domains of the M1 RNA, the evolution of
RNase P and the role of the -1 residue in the mechanism of RNase P RNA
mediated cleavage.
In my study I used the M1 RNA and Pyrococcus furiosus (Pfu) RNase P,
with and without the protein subunite, as model enzymes. Various tRNAderived model substrate was used as the model substrates (See before).
30
Induced fit model
There are several models in enzymatic catalysis to describe the procedure of
how an enzyme interacts with its substrate, binds it, and eventually releases
the product. The induced fit model, the lock and key model, the Concerted
model and Sequential model are some of them. The lock and key model explains the process as one where the enzyme and substrate form a complex
from rigid complementary geometries, like a lock and a key. In contrast, the
induced fit model states that the binding of the substrate slightly changes the
conformation of the flexible active site of the enzyme and eventually leads to
a better binding and catalysis (Figure 9). In the Concerted model and the
Sequential model the structure of the enzyme changes after the binding of
the substrate. In the Concerted model, the change is caused by the same substrate, but in the Sequential model the change of one subunit favors the
change of another subunit.
substrate
The conformation
of the active site
changes slightly to
fit the substrate
Active site
Substrate is
going to bind on
the enzyme
Substrate binds
to the Enzyme
The Enzymesubstrate complex formed
Products are
released
Figure 9. Elucidation of the Induced fit model. The large, lower darker half circle
indicates the enzyme and the lighter, upper form indicates the substrate. The color
change of the substrate indicates the conversion of substrate to product. The figure is
modified from Wikipedia (http://en.wikipedia.org/wiki/Induced_fit#Induced_fit).
The selection of the aminoacyl-tRNAs by the ribosome was suggested to
follow the induced fit mechanism. Using kinetic studies, it was found that
the codon-anticodon recognition not only stabilized the aminoacyl-tRNA
binding on the ribosome, but also stimulated the forward reaction of the pro31
ductive pathway (Rodnina 2001). The hammerhead RNA is also suggested
to use the induced fit mechanism. The Schistosoma mansoni hammerhead
structure has a peripheral loop-bulge structure that can enhance the catalytic
rate from a distal interaction (Nelson 2006; Martick and Scott 2006).
An induced fit mechanism also has been suggested in RNase P mediated
cleavage (Guerrier-Takada et al. 1989). In this study the residue number 92
was deleted in M1 RNA which resulted in a mutated M1 RNA with an altered cleavage site on the mutated substrate that lacked CCAUCA sequence
at the 3’ end. This altered cleavage can be reversed by adding C5 (the protein part of the E. coli RNase P) or 3M ammonium, which indicates that the
structure of the enzyme-substrate complex is flexible.
Investigation in Paper I & Paper II
Previous studies have shown that the TSL-region binds to the residues in the
S-domain (TBS-region), and that changes in the TBS-region of B-type
RNase P RNA affect substrate binding. Therefore, in paper I and II we
wanted to investigate whether changes in the TBS-region in a type A RNase
P RNA affect cleavage on the substrates that have various numbers of RNase
P determinants.
In paper I and II, our data suggest an induced fit mechanism in M1 RNAmediated catalysis (paper I and paper II). We found that the productive TSL/ TBS interaction can change the conformation of the M1 RNA-substrate
complex and results in a distal influence on the events at the cleavage site. It
is noteworthy that the effect is decreased at higher Mg2+ concentration. This
emphasizes the importance of Mg2+ as well as the flexibility of this interaction. This observation has been further confirmed by experiments in paper II.
In order to create a conformational change at the TBS region in M1 RNA,
we used various mutations (or reverse) on the base pair G125/C235 that is
located in the TBS region in M1 RNA. We used Pb2+-induced cleavage to
confirm the conformational change in TBS region. As shown in the paper I,
we obtained a change in the TBS region after we disrupted or reversed the
G125/C235 base pair. When we increased the concentration of Mg2+ in Pb2+induced cleavage, the cleavage pattern of mutant G235 was changed. This
indicates that the base pair of G125/C235 is important for metal ion binding.
In paper I and II, we used substrates with different structures and this allowed us to analyze the interactions between the TSL and TBS regions, as
described above. To date, C-1/G+73 pairing leads to miscleavage at the -1
residue, which is one residue before the canonical cleavage site (Pettersson
and Kirsebom 2008). It has also been suggested that a change at the -1 position disturbs the +73/294 interaction (see above). Therefore we used sub32
strates with mutations at the -1 residue to analyze the effect of the TSL/TBS
interaction on the cleavage site. Note that all the alternative cleavage sites
different from the canonical cleavage site, are consider as miscleavage.
Because of the distinctive behavior of pATSerCGGAAA that we found in paper I, we decided in paper II to investigate the impact of the tetraloop GAAA.
The tetra loop sequence GNRA that the model substrate pATSerCGGAAA has
is well defined in structure (Heus et al. 1991). It is a stable construct and
distinguishable from other tetra loop structures. pATSerCGUUCG and pATSerCGCUUG are two substrates derived from pATSerCGGAAA (see description
above). These two tetraloop structures have been experimentally determined
in the last decade (Jucker & Pardi 1995; Ennifa et al. 2000). We made these
two tetra-loop substrates to define the effects of the tetra-loop structure in
the cleavage site selection and rate of cleavage (see paper I, II III and IV; see
above).
Cleavage site selection
We observed in paper I and II that an altered TSL region in the substrates
resulted in miscleavage. The pTS-L and pATSer substrates have the intact Tstem/loop structure, meaning that the TSL/TBS interaction is not disturbed,
the M1 RNA and all the M1 RNA mutants can still cleave the substrates at
the canonical cleavage site. But interestingly for pATSerCG: the -1 cleavage
frequency went up as the Mg2+ concentration was raised. pATSerCGGAAA has
a disrupted TBS/TSL interaction, therefore the M1 RNA cleaved this substrate mostly at the -1 position. In the case of pATSerUGGAAA, the TSL/TBS
interaction has been disrupted, but the cleavage site is still exposed because
U-1 cannot pair with G+73 (see above and Pettersson et al. 2008), that is
most likely the reason why this substrate does not give miscleavage. Like
pATSerCGGAAA, the other two tetraloop substrates all had miscleavage at -1
position (paper II), but the -1 cleavage frequency was much lower than for
pATSerCGGAAA. The most interesting substrates are the pMini3bp substrates.
They do not have the T-stem/loop at all. This means that the TSL/TBS interaction is lost when these substrates are used. Therefore the impact of the
TSL/TBS interaction on the cleavage site is lost as well. So as we observed
in the experiment all the M1 RNA derivatives could still cut the pMini3bp
substrates mostly at the right position, as in the case of the full length tRNA
precursor.
We also saw that changes in the TBS region of the M1 RNA rescue the miscleavage caused by all three tetra-loop substrates. This suggests that the TSL
region (the T-loop structure) influences the cleavage site selection. This, and
the comparison of the cleavage of M1 RNA with G235 on all tetraloop substrates and pATSerCG suggests that the topography in the vicinity of TBS
region affected cleavage site selection as well.
33
Mg2+ profile
To date, we have found that decreasing the size of the substrates results in an
increase in the requirement of Mg2+. For example, the pMini3bp substrates
required more Mg2+ than the others.
From the Mg2+ curves we also calculated the Hill coefficient of the substrates in paper II (about the Hill coefficient see above). The Hill coefficient
of pATSerCGGAAA was much higher compared to other tetraloop substrates
and pATSerCG, especially at the -1 cleavage site. Apart from pATSerGAAA,
the G235 mutant increased the Hill coefficient for the rest of the tetraloop
substrates. Note that in paper III we found that the Pfu RNase P RNA (the
TBS region is different compare to that in M1 RNA) gave the same Hill
coefficient as G235 with tetraloop substrates.
These data show: 1) The Mg2+ stabilizes the RNase P RNA-substrate complex, helping the RNase P RNA to cleave its substrates. 2)The lower the
number of determinants, the higher the number of Mg2+ ions that are required
for cleavage. 3) The loop structure affects the Mg2+ binding site and therefore influence the RNase P RNA-substrate complex stabilization. 4) The
structure of the T-loop affects the critical Mg2+ binding sites for +1 and -1
cleavage sites differently. 5) Changing the structure of the TBS region influences the number of critical Mg2+ binding sites, and thereby influences the
stability of the RNase P RNA-substrate complex.
Cleavage rate
To further analyze the mechanism of catalysis, we determined the kobs and
kobs/Ksto for all the substrates under single turn over and pH 6.1 conditions
(see above about the kinetics). To date, we found: 1) The fewer the number
of determinants that the substrate has, the less efficiently the M1 RNA
cleaves it. The pMini3bp substrates had the lowest kobs; pTS-L and pATSer
were the fastest substrates. 2) The C-1 substrates had lower efficiency compare to the U-1 substrates. This might be due to the exposure of the cleavage
site. In other words, if the cleavage site is open (as is the case in the U1/G+73), the M1 RNA cleaves more efficiently. For example, pATSerUG is a
faster substrate than pATSerCG. 3) Productive TSL/TBS interaction influences the rate of cleavage. This was seen in the cases of pTS-L and pATSer
derivative substrates that were cleaved by the M1 RNA mutants - the rates of
cleavage decreased. In the case of pMini3bp substrates, however, the mutants of the M1 RNA did not lower the efficiency of cleavage. This is the
same as when pATSerGAAA substrates were used. 4) The loop conformation
influenced the cleavage rate of RNase P RNA at the cleavage site. The pATSerCGUUCG and pATSerCGCUUG were much slower substrates for M1 RNA
compared to pATSerCG and pATSerCGGAAA. 5) Changes in the TBS region
34
could also influence the cleavage rate and binding affinity. This was seen by
comparing M1 RNA to G235 cleavage of the tetraloop substrates.
Note that in paper II we used the optimal amount of Mg2+ in determining the
rate of cleavage. This was not the case in paper I. For example in paper I we
observed that pTS-L(U-1) and pATSerUG had different rates at low Mg2+
concentration, but the difference became much less when the Mg2+ concentration increased. This is probably because we did not use the optimal
amount of Mg2+ in those experiments. The optimal (approximately) amount
of Mg2+ (according to the Mg2+ profiles that we had for all the RNase P
RNA- substrate combinations) allowed us to compare the results in all the
RNase P RNA-substrate combinations without a risk of bias caused by the
different Mg2+ requirements (see above and paper I & II).
Binding constant
The binding constant appKd differed when the structure of the substrate or
the -1 residue identity changed. To date, disturbing the TSL/TBS interaction
increases appKd. However, for the substrates that lack the interaction with
the TBS region, structural changes of the TBS region do not make significant differences on the binding constant. Moreover, changing the -1 residue
from U to C also increased the binding constant, but these increases were
reduced when the divalent metal ion concentration was increased. This
shows the importance of the divalent metal ion in the RNase P RNA mediated catalysis. For further information, I refer to Kikovska et al. 2006, 2005;
Zahler et al. 2003; Loria et al. 1997.
Mn2+ and cleavage site selection
Mn2+ can also induce RNase P RNA cleavage (Brännvall et al. 1999). It can
cause miscleavage at -1 when pATSerCG is cleaved by the M1 RNA wild
type. However, in our experiments we observed that the G235 mutant can
rescue the miscleavage. This strongly indicates that a change in the structure
topography of TBS region influences the position of divalent metal ion(s) in
the vicinity of the cleavage site. This might also be one of the reasons why
the miscleavage was recovered when the structure of the TBS region was
changed.
Alterations in and at the vicinity of the cleavage site
Previous data have shown that changing U to C at position 294 in M1 RNA
(the U294/ +73G interaction, part of the RCCA/ P15 interaction, Fig. 6)
could make the effect of the C-1/G+73 disruption smaller (Brännvall 1999;
2001). In my experiments I found that C294 actually rescued, to some extent,
the miscleavage caused by the M1 RNA wild type, especially for tetraloop
substrates. C294 also influenced the Hill coefficient of pATSerCG. When it
comes to the rate of cleavage, C294 did not increase the rate of pATSerCG
35
much, but C294 increased the rate of pATSerCG tetraloop substrates at the
+1 cleavage site, but not on the -1. However the binding constant was quite
similar for the M1 RNA wild type and G235 when tetraloop substrates were
used. In conclusion, the data here show 1) Strengthening the +73/294 interaction compensate for a nonproductive TSL/TBS interaction to some extent.
2) The mutation from U to C at position 294 might affect functionally critical Mg2+ binding at the vicinity of the cleavage site. 3) Strengthening the
+73/294 interaction also improves the cleavage rate, especially at the +1
cleavage site. 4). C294 works like a suppressor for cleavage at the -1 site.
We also exchanged the 2’OH to 2’H on residue G+1 in the pATSerCGGAAA
which resulted in lower cleavage frequency on -1 compare to intact pATSerCGGAAA, when cleaved by M1 RNA variants.
In summary
The productive interaction of TSL/TBS influences the cleavage site selection, RNA-substrates binding, Mg2+ requirement and the rate of cleavage.
The evidence indicated that M1 RNA-mediated cleavage can be explained
by the induced fit mechanism. The divalent metal ions, together with the
chemical groups in the vicinity of the cleavage site, also play an important
role. The identity of the -1 residue also contributes to the efficiency of cleavage. G235 is important in the TSL/TBS interaction.
The induced fit model in paper I and II is the first properly investigated such
kind of mechanism in RNase P RNA-mediated catalysis. The RNase P
RNA-substrate-protein complex crystal structure (Reiter et al. 2010) provided new data that allowed us to analyze the Induced fit model in more
detail and get a further understanding of the mechanism. From a close examination of the residues in contact with the TSL region, we suggest that
stacking might be the important mechanism for establishing a productive
TSL/TBS interaction (see paper II). Whether this is also the case for other
types of RNase P RNA remains to be discovered.
36
The two domains of the RNase P RNA
RNase P RNA has two domains, as mentioned before (Figure 5). To date,
the two domains of RNase P RNA fold to their native structure independently of each other (Baird NJ et al. 2005). However, data also showed that
the two domains interact with each other during folding in type A RNase P
RNA. The L8 loop and P4 helix interact with each other and the L9 loop
interacts with the P1 helix (Massire et al. 1998). That says that domaindomain interactions play an important role in the folding of type A RNase P
RNA. These two domains are not only folding independently of each other
as well as cooperatively, they also contribute to the function of RNase P
RNA both independently and cooperatively.
Contribution of the S-domain
We chose Pyrococcus furiosus (Pfu) RNase P RNA in paper III to investigate the impact of the S-domain and the relation between S-domain and Cdomain. Pyrococcus furiosus (Pfu) is an Archaea species. This species is
special because the optimal growth temperature for it is 100°C. At this temperature most of the living organisms would be destroyed. Thus Pfu is classified as a hyperthermophile.
Archaeal RNase P contains one RNA subunit and 4-5 proteins. The RNA
subunit has the catalytic property on its own in vitro (Pannucci et al. 1999;
Tsai et al. 2006; Li et al. 2008).The RNA subunit of Pfu RNase P belongs to
the A-type RNase P RNA, the same type as E. coli RNase P RNA. A comparison of the secondary structures of these two RNAs shows that they have
similar C-domain but different S-domain structure in the P10 and P11, where
the TBS region is located (Gopalan, 2007 and description above). The special properties of Pfu RNase P are that its protein part is homologous to the
eukaryotic RNase P protein (Hall and Brown 2002), but the RNA part is
more like bacterial RNase P RNA that performs catalysis in vitro on its own
at a much higher efficiency than Human’ RNase P RNA (Pannucci et al.
1999; Tsai et al. 2003, 2006).
37
The Pfu holoenzyme has been successfully re-assembled in vitro by Gopalan‘s group (Tsai et al. 2006), providing us with an opportunity to study the
Pfu holoenzyme part by part in vitro.
Present investigation in Paper III
We used Pfu RNase P, both with and without the protein part, to cleave our
model substrates. Pfu RNase P could recognize these substrates and cleave
them in vitro, both in the absence and presence of the protein part.
As mentioned previously, Pfu RNase P RNA has a similar C-domain as E.
coli RNase P RNA (M1 RNA), but the TBS region in its S-domain is different. M1 RNA cleaved the substrate that has a paired cleavage site and a disturbed TSL region mainly on -1, i.e., pATSerCGGAAA. If the TBS region in
the M1 RNA was disrupted, then the miscleavage was recovered. However,
in the Pfu RNase P RNA, the TBS region is different from M1 RNA, and the
substrate pATSerCGGAAA was cut mainly at +1 instead.
However, if we exchanged the S-domain of Pfu with the S-domain of M1
RNA, the newly constructed RPREcS3 cleaved pATSerCGGAAA again mainly
at -1, just like we observed when we used M1 RNA to cleave this substrate.
This interesting observation indicates the importance of the S-domain, especially the TBS region, in the cleavage site selection. The S-domain of Pfu
RPR was suggested to interact differently with the substrate compared to M1
RNA’s S-domain. This also follows the induced fit model that we proposed
in the paper I and II: a change of the TBS/TSL interaction can lead to conformational changes of the RNase P RNA-substrate complex and from distal
disturb the happening at the cleavage site. A similar observation was made
by replacing Methanothermobacter thermautotrophicus RNase P RNA’s Sdomain with M1 RNA’s S-domain. This resulted in an improvement in the
catalytic activity (Li, 2008). The improvement might also be due to improved substrate binding by using the M1 RNA’s S-domain.
The interesting aspect of the Mg2+ profile is that pATSerUG required more
Mg2+ when it was cleaved alone by Pfu RPR, compared to when the M1
RNA was used. The pATSerUG and pMini3bpUG had very similar Mg2+
requirements, which was the opposite for to M1 RNA induced cleavage.
Pfu is a hyperthermophile, so we determined the cleavage rate of Pfu RNase
P RNA on the model hairpin substrate both at 37 C and 55 C (55 C is the
optimal cleavage temperature for Pfu RNase P RNA). Pfu RNase P RNA is a
slower enzyme compared to M1 RNA. Pfu RNase P RNA gives faster cleavage at 55 C, which is consistent with the observation that was made with for
Thermus therimophilus RNase P RNA (Hartmann, 1991). There was almost
38
no difference in rate and dissociation constant Kd for pATSerUG compared
to pMini3bpUG when cleaved by Pfu RNase P RNA.
The protein subunit of Pfu consists of 4 proteins that form two binary complexes: POP5•RPP30 and RPP21•RPP29. The footprint experiment showed
that the RPP21•RPP29 binary complex might affect the TSL/TBS interaction
or it might actually bind to the TSL region of the substrate (Xu, et al. 2009).
Our experiments showed that when Pfu RNase P RNA cleaved the substrate
in the presence of RPP21•RPP29 at a lower Mg2+ concentration, it behaved
like the wildtype M1 RNA. However, when the Mg2+ concentration increased, the miscleavage was recovered, like the case with only Pfu RNase P
RNA. The POP5•RPP30 was suggested to bind on the C domain of Pfu RPR
(Tsai, et al. 2006; Xu, et al. 2009). In contrast to RPP21•RPP29,
POP5•RPP30 did not make any significant change on Pfu RNase P RNA
cleavage site selection. The holoezyme behaved in the same way as
RPP21•RPP29+ Pfu RNase P RNA with respect to cleavage site selection.
All these data indicate that 1) The TSL/TBS interaction is important for
cleavage site selection and the RPP21•RPP29 significantly influences on the
cleavage selection. 2) Mg2+ plays an important role in the interaction. 3) This
is further support for previous investigation that found that the C-1/G+73 pairing in pATSerCGGAAA influences the cleavage site selection.
In summary
1) The S-domain was important for substrate binding, cleavage rate and
cleavage site selection (also see Brännvall et al. 2007). 2) The S-domain
from Archaeal RNase P RNA interacted differently with the substrate compare to the bacteria one. 3) The Pfu RNase P protein complex RPP21•RPP29
could alter this difference. 4) The TBS/TSL interaction was important for
cleavage by disturbance from distal of the cleavage site conformation or by
disturbance of the metal ions or chemical groups in the vicinity of the cleavage site. 5) The C-1/ G+73 could lead to miscleavage. 6) POP5•RPP30 interacted with the C-domain of the Pfu RPR and only led to miscleavage on the
substrates that had only RCCA-/RNase P RNA interactions. 7) The positioning of the divalent metal ion was very important for the cleavage. The data
suggested that changing the positioning of the divalent metal ion led to low
efficiency cleavage and incorrect cleavage.
39
Contribution of the C-domain
The catalytic domain (C-domain) is considered the ancient part of the RNase
P RNA (Sun, et al. 2010). The C-domain can perform catalytic activity in
vitro without the S-domain, in the absence and presence of the protein part
(Green and Vold, 1996; Loria and Pan, 1999; Kikovska et al. 2011). Therefore the C-domain might have existed first as a catalytic RNA. Then, as requirements evolved, like higher rate and complexity in substrate selection,
the S-domain evolved, followed by the evolution of the protein part of
RNase P. There exist small RNase P RNAs in nature which resemble the
secondary structure of the C-domain. For example, the Saccharomyces cerevisiae mitochondrial RNase P RNA does not have the corresponding Sdomain nor the P15-17 region (Seif et al. 2003), but this RNase P RNA has
no catalytic activity without the protein part of the RNase P RNA in vitro.
However, there are two other mini RNase P RNAs from Caldivirga maquilingensis and Vulcanisaeta distribute (Lai, et al. 2010). These two mini
RNase P RNAs lack the structure corresponding to the specificity domain
and they can actually act in trans, in vitro on the substrates without the help
of the protein. The existence of these small RNase P RNAs in nature provides evidence that the C-domain most likely was the first catalytic part of
RNase P RNA that evolved in nature.
Present investigation in Paper IV
In paper IV we wanted to investigate the impact of the C-domain in catalysis
by RNase P RNA. In our experiments we observed the catalytic activity in
vitro of the C-domain construct (CP RPR) on the model substrates (see paper
II and IV and descriptions above).
The CP RPR cleaved all the tetraloop substrates at the canonical cleavage
site irrespective of the concentration of Mg2+, which is in contrast to our
observations using M1 RNA (see paper I, II). This was expected because the
C-domain does not have the TSL/TBS interaction with the substrates, as in
the case of M1 RNA cleavage of pMini3bp substrates (see paper I and IV).
The S-domain therefore influences the cleavage site selection. Without the
TBS region interacting with the TSL region, the RNase P RNA generates
much less miscleavage on its substrate. This again provides a link between
TSL-/TBS and events at the cleavage site.
Looking at the Mg2+ profile, we observed that there was almost no difference
in the Mg2+ requirement for pATSerUG and pMini3bpUG when CP RPR
was used, which is in contrast to M1 RNA (see paper I). We also calculated
the Hill coefficient. Table 2 shows that pMini3bpUG required more Mg2+
than pATSerUG during the cleavage of M1 RNA. We observed the same
40
effect when pATSerCG and pATSerCGGAAA were cleaved by CP RPR and
M1 RNA: they both need higher amounts of Mg2+ when cleaved by CP RPR,
but there was no difference between them when cleaved by CP RPR (Table 2,
taken from Wu et al. manuscript). These observations also reflect the importance of the TSL/TBS interaction. When the S-domain is deleted, more Mg2+
will be needed to stabilize the RNase P RNA-substrate complex. The base
pairing of C-1/G+73 also increases the Mg2+ requirement.
Earlier studies have suggested that Mn2+ can coordinate differently in the
vicinity of the cleavage site resulting in increased miscleavage frequency at
the -1 position (Brännvall and Kirsebom 1999; 2001; Brännvall et al. 2002,
2003 and 2007; Kirsebom 2007). However, when CP RPR was used, the
miscleavage at -1 did not increase, suggesting that the TSL/TBS interaction
influences the positioning of the functional divalent metal ions at and in the
vicinity of the cleavage site.
Table 2. Hill coefficients of different substrates with M1 RNA and C-domain
M1 RNA
CP RPR
pATSerUG
pMini3bpUG
+1
+1
2.1 ± 0.023
3.2 ± 0.19
3.3 ± 0.5
3.2 ± 0.33
pATSerCG
+1
3.4 ± 0.46
2.4 ± 0.1
pATSerCGGAAA
-1
1.4 ± 0.39
+1
3.7 ± 0.37
-1
4.6 ± 0.24
2.8 ± 0.3
CP RPR gave a much lower rate of cleavage compared to a full-length
RNase P RNA. The dissociation constant Kd also dropped when the Cdomain was used compared to M1 RNA. However there were no significant
differences between pATSerUG and pMini3bpUG in the rate and Kd value
when CP RPR was used, which is different from M1 RNA-mediated cleavage. The similar effect was seen with pATSerCG and pATSerCGGAAA. The
data above suggest that the presence of the S-domain increases the binding
affinity and rate of cleavage. Nonethelss, when the C-1/G+73 kind of substrates were cleaved by CP RPR, they gave significantly reduced rates compared to M1 RNA. A similar phenomenon has been seen in type B RNase P
RNA mediated cleavage as well (Loria and Pan, 1999). These observations
bring up an interesting interpretation that the result of the evolution of the Sdomain is to increase the rate of cleavage, in particular by suppressing the
negative influence of the C-1/G+73 base pair on the cleavage efficiency. At the
same time the accuracy of cleavage is lost.
41
In our experiments we found that changing the U294 to C294 in M1 RNA
resulted in a rate increase when pATSerCG and pATSerCGGAAA were used
(paper II). This was observed in CP RPRC294 induced cleavage as well, but
the rate increase of tetraloop substrate cleavage with CP RPRC294 is not as
great as during the cleavage with the full-size M1 RNA. This result suggests
that the +73/294 interaction plays a role in cleavage efficiency. It also indicates that TSL/TBS interaction might contribute to breaking the C-1/G+73 pair
in the substrate when the RNase P RNA-substrate complex is established
(also see paper I and II).
When we compared the substitution of A248 with G248 in M1 RNA with
CP RPR mediated cleavage, we observed something different. The M1
RNAG248 cleaved pATSerCG at an increased rate at -1 compared to wild type
M1 RNA, while the rate at +1 remained unchanged. The Hill coefficient
changed slightly for M1 RNA wild type and M1 RNAG248. We also observed
that the M1 RNAG248 binds pATSerCG with lower affinity compared to M1
RNA wildtype. This was not the case when CP RPR was used in cleaving
pATSerCG. The rate of cleavage by CP RPRG248 actually increased significantly compared to wildtype CP RPR as did the Hill coefficient when CP
RPRwt was mutated to CP RPRG248. This could indicate that the N-1/G248 interaction influenced the charge distribution in the vicinity of the cleavage
site. This in turn affected the positioning of the divalent metal ion and as a
result, the cleavage rate for CP RPRG248 increased compare to CP RPRwt.
These data together emphasize the coupling of the TSL/TBS interaction and
the events in the vicinity of the cleavage site.
In summary
1) Deleting the S-domain resulted in less efficient cleavage. 2) Absence of
the S-domain resulted in higher accuracy in the cleavage. 3) The structural
architecture of the -1/+73 pair is important for the cleavage, for both the rate
and the cleavage site selection. 4) A productive TSL/TBS interaction can
help to open the C-1/G+73 by influencing the N-1/A248 and +73/294 interactions. 5) The results in this paper support the induced fit model.
42
What is happening around the cleavage site?
RNase P cleaves its substrates at the canonical cleavage site. Studies around
the cleavage site have always been one of the big focuses in RNase P research. Those studies include: the interactions in the vicinity of the cleavage
site; the residues in the vicinity or at the cleavage site and chemical groups
and critical Mg2+ binding sites around the cleavage site.
There are a number of interactions, between RNase P RNA and pre-tRNA
near or at the cleavage site: the RCCA-/ P15 loop interaction; the -1/A248
interaction; and the +73/U294 interaction (see introduction above). The protein part of bacterial RNase P was suggested to interact with the 5’ leader
sequence of the pre-tRNA (Niranjanakumari et al. 1998). The -1/ A248 interaction was reported by Zahler and colleagues in 2003. They found that mutating A248 can lead to miscleavage of substrates that have 2’H at N-1. Specifically, they showed that U248 can lead to a miscleavage of the pre-tRNA
when the -1 reside is deoxy U, but that this miscleavage can be restored if
the -1 residue is deoxyA. The same observation was also observed on the
RNase P holoenzyme reaction. It was shown that the A248 binding defect
can also be recovered in this way. They proposed that the U-1/A248 interaction is common and important for RNase P mediated catalysis (Zahler et al.
2003). However our data are not consistent with the idea that A248 interact
with residue -1 through WC base pairing (see below).
The C+1/G+72 base pairing in the tRNA precursor cleavage site is believed
to be important for the binding and rate of RNase P RNA-mediated cleavage
(Kikovska et al. 2005). Ema Kikovska and colleagues suggested that the
exocyclic amine (2NH2) of G+1 affects the rate of cleavage. Absence of the
2NH2 affects the charge distribution and positioning of the Mg2+ binding site
in the vicinity of the cleavage site. They also found that the base of the +1
residue is not absolutely necessary, although its presence suppresses the
miscleavage and significantly increases the rate of the cleavage (Kikovska et
al. 2006).
The -2 residue in the tRNA precursor has also been suggested to play a role
in both the cleavage site recognition and the rate of cleavage (Brännvall et al.
1998). In Brännvall’s work, they used M1 RNA and Mycoplasma hyopneumoniae (Hyo P) RNase P RNA, a type B RNase P RNA. They found that
43
these two RNase P RNAs behaved differently when the identity of the -2
residue changed in the tRNA precursor.
The contribution of the -1 residue in the substrate
About 67% percent of the -1 residue in E. coli tRNA precursors are uridines
(Yuriko et al 1990, Kufel and Kirsebom 1996). If the -1 residue is C and can
pair with G+73, this most likely leads to miscleavage at the -1 position, both
in vitro and in vivo (Pettersson and Kirsebom 2008). Data in paper I also
showed that C-1/G+73 substrates need more Mg2+ than U-1/G+73 substrates to
reach the optimal cleavage. In addition, C-1/G+73 substrates are usually slower than U-1/G+73.
Previous work has established the importance of the -1 residue in the tRNA
precursor. The cleavage site selection and kinetics of cleavage are dependent
on the identity of N-1 (Brännvall et al. 2002; Zuleeg et al. 2001). Brännvall
and colleagues found that it is important that the -1 residue is a U nucleotide
(it is also the most common -1 residue in the tRNA precursor), indicating
that it is a positive determinant for RNase P cleavage. On the other hand,
Zuleeg and colleagues used a mini model substrate and they mainly determined the rate of cleavage. Changing the N-1 residue in a full-length pretRNA also leads to miscleavage and a change in the the rate of catalysis by
the RNase P holoenzyme (Crary et al. 1998; Loria et al. 1998; Loria and Pan
1998).
The 2’OH at the -1 residue is also important for the rate of cleavage (Smith
and Pace 1993; Loria and Pan 1998, 1999). Pan and colleagues showed that
a 2’H mutant influenced the rate of cleavage on both correct and incorrect
sites. Brännvall and others also made a change on the -1 residue by replacing
the 2’OH with 2’NH2. They found that the 2’N at -1 influenced cleavage-site
recognition, substrate binding, and rate of cleavage. They also suggested that
the 2’OH of the -1 residue is important for the charge distribution in the vicinity of the cleavage site, as the result influences the positioning of the divalent metal ion (Brännvall et al. 2003, 2004; Persson et al. 2003).
Present investigation in Paper V
For all the reasons above, we decided to investigate the impact of residue -1
in the tRNA precursor. In particular, we wondered why U-1 makes a faster
substrate than the other variants in E. coli.
C-1/G+73 substrates were slower than U-1/G+73 in our experiments with the
model hairpin structure substrates, but not with the full tRNA precursor
(Zahler et al. 2003; paper I). This raises the possibility that with an increas44
ing number of determinants, the difference of C-1/G+73 and U-1/G+73 can be
masked. To investigate the impact of the -1 residue, we decided to use the
pMini3bp variant substrates that only carry the determinants around the
cleavage site. This means the influence from the TSL/TBS interaction is
removed (see above).
We made a substrate based on the pMini3bp substrate structure but without
the base attached on the ribose at the -1 residue; we called this pMini3bp /G.
This substrate could be cleaved by M1 RNA in our experiment, but it gave
about 50% miscleavage on the -1 residue. The Mg2+ profile of pMini3bp /G
was almost the same as that of pMini3bpUG, but the rate of cleavage
dropped dramatically when the base was deleted. All these data indicate that
the base on the -1 residue is not absolutely needed for cleavage, but its absence leads to miscleavage and serious drop in rate.
As mentioned before, U-1/G+73 is a faster substrate than C-1/G+73. To
avoid the impact of C-1/G+73 pairing, we also compared pMini3bpUA and
pMini3bpCA substrates. Data showed that both substrates were almost always cleaved only at the canonical cleavage site; pMini3bpUA was faster
under single turn over condition compared to pMini3bpCA, regardless of the
Mg2+ concentration. This result is in agreement with the multiple turnover
measurement that was obtained by Brännvall (Brännvall et al. 2003). The
results above indicate that U at the -1 residue makes a faster substrate than C.
O4 in the base of U-1 contributes to the cleavage, but not significantly. (see
Figure 10a)
To further investigate the O2 in the base of U-1, we made the pMini3bpIsoCG substrate. As Figure 10a shows, IsoC is derived from a cytosine.
The difference between IsoC and U is that U has an oxygen at position 2, but
IsoC has an NH2 group at this position. pMini3bpIsoCG could be cleaved by
M1 RNA and usually only at +1 cleavage site, regardless of the concentration of Mg2+. The Mg2+ profile of this substrate was the same as for the other
pMini3bp substrates, but the rate of cleavage was much lower compared to
pMini3bpUG. The binding constant, however, increased somewhat compared to pMini3bpUG. These results indicate that the O2 contributes significantly to the cleavage and should be considered to be a positive determinant
for the M1 RNA mediated catalysis.
45
a
4
3
5
2
6
1
b
Purine
Inosine
Guanine
2DAP
Adenine
DAP
Figure 10. Different base structures used in our experiments. a). Shows the difference of C and U and Iso C. The numbers on the base ring indicate the base numbering. b). Shows the different varieties of purine. The arrows show the difference of
the NH2 group on the molecular structure and the effect that we observed in our
experiments.
In E. coli, about 13% of tRNAs have A as the -1 residue and 8% have G as 1. Therefore, we wanted to investigate the effect of purine as a base at the -1
residue. We used 6 different pMini3bp substrates with purine base at -1 position for cleavage of M1 RNA (See Figure 10b). These substrates all needed
high concentrations of Mg2+ for the cleavage and the rates were much slower
46
than for substrates with pyrimidine at -1. As Figure 10b shows, we compared the rate of different purine substrates. The data indicate that NH2 at
position 2 on the purine base has a negative impact on rate, but no effect on
the binding constant. The NH2 at position 6 does not affect the rate. pMini3bpGG, pMini3bp2APG and pMini3bpDAPG gave miscleavage, but not
the other substrates. These data indicate that NH2 at position 6 does not have
much effect on cleavage selection.
We also used the protein part of the E. coli RNase P (C5) to investigate the
impact of C5 on the cleavage. To date, with help of C5, pMini3bpGG abolished the miscleavage. This implies that C5 acts as a positive determinant
that can rescue the miscleavage caused by the 2NH2. It is worth noting that
G-1 is not pairing with G+73 in the substrate that we were using.
In previous studies, U-1 and A248 were believed to form a base pair with
each other (Zahler et al. 2003). In the RNase P-substrate co-crystal structure
A248 is located close to the 5’ termini of the tRNA (Reiter et al., 2010).
However, we still cannot conclude if U-1 and A248 interact with each other
because the crystal structure represents the post cleavage state, not the transition state interaction. To investigate if the U-1 is forming a WC base pair
with A248, an internal modification has been made on position 3 of the base
U on -1 residue (Brännvall and Kirsebom, 2005). To date, together with the
fact that C-1/G+73 has increased miscleavage frequency when A248 is
changed to G248, we conclude that most likely U-1 and A248 do not interact
via WC base pairing. It has also been suggested that the Hoogsteen surface
of A248 is important in the interaction with the substrate (Siew et al., 1999).
However, our experiments show that this is still unlikely because the cleavage rate did not increase when we replaced the A248 with G248 on cleavage
of -1pMini3bpIsoC (Figure 11). This result indicates that -1U is not base
pairing with A248. The Mg2+ stabilizes the RNase P RNA-substrate complex
and the O2 (and NH2 in G-1) in the base of the -1 residue perhaps involved
in Mg2+ binding. This, together with the Mg2+ profiles, lid us to propose that
the identity of residues or chemical groups in the vicinity of the cleavage site
influences the charge distribution and thereby disturbs the positioning of the
divalent metal ion, consequently influencing the rate and accuracy of cleavage. This model is also supported by Kikovska’s result (Kikovska et al.
2006).
In conclusion, in paper V we found: 1) The presence of the base in the -1 is
not essential for M1 RNA mediated cleavage. 2) The two oxygens in the
base of U-1 contribute to the cleavage; in particular, O2 contributes the most
to the catalysis. 3) Purines as bases at -1 residue promote in poor cleavage
compare to pyrimidine. 4) 2NH2 on the purine base is a negative determinant, both for cleavage accuracy, and efficiency of cleavage at the canonical
cleavage site. 5) Residue at -1 is likely not involved in WC basepairing with
47
A248. However, the identity of the residue in the vicinity of the cleavage site
influences the charge distribution and thereby influences the position of the
functionally important Mg2+.
Figure 11. Schematic diagram of possible -1U/A248 interaction through Hoogsteen
edge. The dark circle indicates the residues that are involved in the Hoogsteen edgeinteraction.
48
Concluding remarks
The work that I present in this thesis contributes in understanding according
to the following:
1) The importance of the base in the -1 residue in M1 RNA mediated cleavage. The base in the -1 residue is not absolutely necessary, but its absence
causes miscleavage on the -1 cleavage site. The O2 in the Uracil base contribute positively to the cleavage rate. The purines are not good for RNase P
RNA mediated cleavage. 2NH2 influences the cleavage negatively. The -1
residue is not pairing with A248. The identity of the -1 residue influences the
charge distribution, therefore influences the Mg2+ positioning and the cleavage.
2) Functional coupling between the TSL/TBS interaction and the events at
the cleavage site. The TSL/TBS interaction affects the cleavage site selection,
cleavage rate and ground state binding. It can also affect the position of Mg2+
in or at the vicinity of cleavage site. G235 in M1 RNA is important in the
TSL/TBS interaction. C-1/G+73 pairing affects the cleavage site selection.
Finally we suggest that stacking plays an important role in RNase P RNAsubstrate interaction.
3) Contribution of the S-domain to the cleavage. The S-domain is important
for the cleavage rate, the cleavage site selection and the substrate binding. It
increases the cleavage rate but decreases the accuracy of cleavage site selection. The S-domain of Pfu RPR interacts differently from that of M1 RNA.
Constructs that contain E. coli S-domain and Pfu C-domain have a cleavage
site selection similar to that of E. coli RNase P RNA (M1 RNA). Pfu
RPP21•RPP29 binds on the S-domain of Pfu RNase P RNA and confers the
Pfu RNase P RNA with a cleavage site selection similar to M1 RNA. The
POP5•RPP30 binds on the C-domain of Pfu RNase P RNA and leads to miscleavage when the only determinants are in vicinity of the cleavage site.
4) Contribution of the C-domain. Without the S-domain, C-domain gives
more accurate, but slower, cleavage. This is important for understanding the
evolution of the RNase P RNA. Without the TSL/TBS interaction, there is a
higher requirement of Mg2+ in substrate-enzyme stabilization. The +73/294
interaction is important for cleavage efficiency. The N-1/248 interaction influences the positioning of the divalent metal ion and cleavage efficiency.
49
G248 especially influences the charge distribution and results in repositioning of the divalent metal ion.
In our study we compared the Mg2+ requirement, cleavage site selection, and
cleavage rate of different substrate-enzyme combinations to study the mechanism of RNase P mediated cleavage. The RNase P-substrate cocrystal
structure solved in 2010 (Reiter. et al. 2010) has contributed significantly to
the research on RNase P. The combination of biochemical evidence and
direct evidence, perhaps from evolving methods in biochemistry and structural biology will provide us with more information to eventually solve the
mechanism of RNase P mediated cleavage. For example, in studying the
transition state cleavage, the developing non-crystal-dependent femtosecond
X-ray method may be useful.
One of the most important tasks of biology is to cure disease and prolong
human life. To this end we are trying to find drugs that can target disease but
do not damage the cell in the process. RNase P is a perfect potential drug
target, for two reasons. First, RNase P is indispensable for bacterial viability
(Schedl et al. 1974; Wegscheid and Hartmann 2006; Wegscheid et al. 2006;
Kobayashi et al 2003). Second, the architecture of RNase P differs in Bacteria and Eukarya. For example, the loop (p15 loop) that is missing in Eukarya
has a significant role for Bacteria, making it a good target for drug design.
There are several approaches that have been developed to design drugs targeting RNase P. For example aminoglycosides has been found to inhibit the
catalytic activity of RNase P. Hopefully some of the knowledge we get today
can be used as tools to solve health problems in the future.
50
Swedish Summary
RNas P är ett ribonukleas som klyver bort den extrasekvens som finns i 5’änden av omogna tRNA-molekyler. RNas P är essentiellt och har hittats i
alla levande organismer. Det består (med enstaka undantag) av minst en
RNA-komponent (RPR) och en proteinkomponent (RPP). Antalet proteinkomponenter varierar beroende på ursprunget hos RNas P. I bakterier finns
endast ett RNas P-protein, medan det finns fyra till fem olika proteiner i
arkéer och minst nio hos människor. Det är RNA-komponenten som står för
den katalytiska aktiviteten (förutom i ett fåtal fall där RNas P enbart består
av protein) och kan klyva omogna tRNAn in vitro (i provröret) i frånvaro av
proteinkomponenten. Därför är RNas P ett ribozym. Inuti levande organismer, in vivo, är dock proteinkomponenterna livsnödvändiga.
Eftersom RNas P är absolut nödvändig i cellen är det av vikt att förstå mekanismen för RNas P-medierad katalys. Detta kommer att leda till en allmän
förståelse av ribozymkatalyserad klyvning och evolution av RNA-molekyler.
Dessutom kan kunskap om RNas P leda till att vi kan utveckla nya läkemedel mot exempelvis olika bakterierelaterade sjukdomar. Under tidigare årtionden har framsteg gjorts som gett oss förståelse för hur RNas P:s struktur
ser ut, för hur den viker ihop sig från en endimensionell sekvens till sin slutliga tredimensionella form, för hur RNas P känner igen och binder till sina
substrat, för vilken betydelse tvåvärda metalljoner har för klyvningen, för
vilken mekanism och vilken kinetik som kännetecknar katalysen, och för hur
RNas P har utvecklats sett från ett evolutionärt perspektiv. Trots att mycket
redan är känt finns flera obesvarade frågor som behöver lösas. Hur ser den
grundläggande kemin bakom RNas P-medierad katalys ut? Vilka nukleotider
eller kemiska grupper i det katalytiska sätets närhet är inblandade i klyvningsprocessen? RNas P interagerar med sina substrat, men hur ser interaktionen ut mellan det att substratet bundits och den klyvda produkten frigörs
och hur påverkar dessa interaktioner katlysen? Vilken konformation har
RNas P i ”transition-state” och hur påverkar denna konformation klyvningen? Exakt vad för roll har de tvåvärda metalljonerna? Hur mycket bidrar
specificitetsdomänen (S) och hur mycket bidrar den katalytiska C-domänen
till katalysen och vilken roll har de haft i evolutionen av RNas P? Vilken
funktion har RNas P-proteinerna?
51
Målsättningen för arbetet som den här avhandlingen baseras på var att förstå
hur klyvnigsmekanismen och substratinteraktionen fungerar i RNas Pmedierad katalys, med fokus på hur de två RNas P-domänerna (S och C)
bidrar till klyvningen, samt vilken betydelse basen i position -1 i substraten
(omedelbart innan klyvningsstället i det omogna tRNA:t) har. Jag har huvudsakligen använt mig av RNA-komponenten hos RNas P i mina studier.
Jag har använt mig av biokemiska verktyg, samt olika konstruktioner och
mutanter av RNas P-RNA och olika modellsubstrat för att få användbar information om katalysen. Vi har jämfört data från olika RNas P-RNAsubstratkombinationer med avseende på t. ex. Mg2+-profiler, val av klyvningsställe, och klyvningshastighet. Med hjälp av dessa data utvärderade vi
sedan olika tänkbara klyvningsmekanismer för RNas P. De olika RNas PRNA jag studerat var RNas P-RNA (RPR) från bakterien Escherichia coli
(M1 RNA) och från arkéen Pyrococcus furiosus (Pfu), med och utan respektive proteiner. Som substrat använde jag mig av olika modellsubstrat baserade på tRNA-molekyler.
Tidigare studier har visat att tRNA redan i omogen form är ihopvikt till sin
karakteristiska form och att RNas P känner igen och klyver den (Chang et al.
1973). TSL-regionen (T-stem-loop) i omogna bakterie-tRNA och TBSregionen (TSL-binding site) i RPR interagerar när RPR-substratkomplexet
bildas. Våra data visar att en produktiv TSL/TBS-interaktion påverkar händelserna vid klyvningsstället genom att påverka hur de inblandade kemiska
grupperna och/eller Mg2+ positioneras så att en effektiv och korrekt sker.
Detta överensstämmer med en klyvningsmekanism som kallas ”induced fit”
(delarbete I och II).
Vidare har våra data visat att den katalytiska domänen (C-domänen) kan
klyva substraten i frånvaro av specificitetsdomänen (S-domänen). Klyvning i
närvaro av S-domänen, som innehåller TBS-regionen, gav en ökad klyvningseffektivitet, men till priset av en försämrad noggrannhet. S-domänen
från Pfu RPR interagerar annorlunda med substraten jämfört med Sdomänen från M1 RNA. Intressant nog så beter sig en hybrid-RPR där Sdomänen från M1 RNA satts ihop med C-domänen från Pfu RPR mest likt
M1 RNA. Vi kom fram till att Pfu-proteiner som binder till S-domänen tar
över RPR:s roll. Detta är viktigt ur en evolutionär aspekt. Klyvningen påverkades också när S-domänen var utbytt eller borttagen vilket överensstämmer
med ”induced fit”-modellen.
Omogna tRNA från Escherichia coli som har en uracil i position -1 är “bättre” substrat än omogna tRNA som har andra baser i den positionen. Basen i 1, och då särskilt karbonylsyret i basens position 2 på U-1, bidrog mest till
effektiviteten och noggrannheten för klyvningsprocessen, och till att stabili52
sera transition state. En purin i position -1, speciellt den exocykliska aminen
i basens position 2, har en negativ inverkan på klyvningen. En möjlighet är
att identiteten av basen i position -1 påverkar laddningsfördelningen och
därmed påverkas positioneringen av Mg2+-jonerna vilket resulterar i ett ändrat klyvningsmönster. Med våra data som bakgrund föreslår vi en konformationsassisterad mekanism som modell för RNas P-medierad klyvning.
53
Acknowledgements
My greatest thanks goes to my advisor, Leif A.K. Without you this work
would never have been possible. I have learned a lot from you, both in science and life. I like the passion that you have in science. I will never forget
you said: Life is a bitch then you die. You should kill the bitch and move on.
This is cool!
Thank you, Anders V. for all the discussions in science. Thank you, Maria
S. Thank you for your great help with my thesis writing and my work! You
are my model of the perfect female scientist. I know now that science and
family can both co-exist in one’s life.
Thank you, people in our group! Ulrika, how can I say thank you adequately! You make me feel warm! Mattias B. thank you to being so patient
teaching me at the beginning; that was important for me. Ema K., thank you
for sharing your knowledge with me. Fredrik P, my personal translator ,
thank you for all your great help in work and thesis writing! I like all the fun
chats! Jaydip G., you taught me a lot! Chen Yu, you are so sweet. Davinanders Väg, Mao Guanzhong, Mohammad.F, Fredrik M., Evgen B.
thank you for being nice and interesting.
Diarmaid H., thank you for teaching me and being so nice. Staffan S. Gerhart W. Santanu D., thank you for being such knowledgeable PI. Nora A.,
thank you for all the chats. Suparna S. and Ray, thank you for your great
help. Chandra S.M. and Ravi K. K., thank you for your great help. Chenhui Huang, thank you for your help. Akiko
You
make me like Japan so much! Eva thanks for making my work so much
easier.
Nils E. M., thank you so much for teaching me and being so patient, and
being a such good person! Urszula thank you for teaching me and the wonderful time! Ulla U. and Hans E, thank you for teaching me and being so
nice; the time in GRENOBLE was wonderful! My special thanks goes to
Terese B., thank you for all the help with my work and thesis! You are so
awesome!
54
Thank you, all my friends! All my Adrenalin Junkies! I have been thinking
how to write this part since last year. I still cannot completely express my
gratitude for having you as my friends! You make me alive, shining, happy,
and give me the courage to keep on going in both science and life; you made
me! Amanda, pity the Italy trip never happened ! Aurelie, I admire you
and I miss all the chats with you! Cia, I wish I were as strong as you (I mean
mentally) ; you make me feel safe! Disa, you make me feel like home!
Emma, not many people understand me as you do! Hava, my dude, you
know what I mean! Marie, my cool idol, how much I wish I could be like
you! Nadja, you little sweetie! Yang, my lovely friend! Fabien, Klas and
Magnus, how lucky I was to be invited to sit in the same office with you! I
enjoyed the fights for the music in the lab, all the jokes you made, all the
loud laughs we had, all the late evenings we spent in the lab, all the after
work late, outside dinners and all the knowledge you taught me! What a
time! Prune, do you really have a tail ? Christina R., you are lovely! Erik
and Johan R., thanks for all the wonderful times! Lei and Jia Mi, thank you
for all the help and chats. Pernilla, Chris T., thanks for all the nice chats.
Henrik T. I like your painting! Hurry, hurry !
Kristin, thank you. Per and Niklas, thank you for all the chats. Rebecka
and Kasia, thanks for the nice teaching and wonderful time. Robert F.
thanks for being such a lovely PI! The most important thing is to be flexible,
Lotta, Sonchita, Johan A., Buppi, thank you. Andrea and Freright?
drik S, thank you for your help. Patricia, Linda, Helien, Pontus, Adam,
Karin T., thank you for the nice time. Petter, Britta, Elin, thank you for
being there. Magnus Lundgren, Mikke and Doreen, thank you. Yanhong
Pang, Kaweng Ieong, thank you for the nice time. Vasili thank you for asking me how is going. Linda and Mattias, it is nice to have you back to
Sweden.
Thank you all my friends outside work. You added so much color in my life!
My parents.
Zhibing, you are special to me. Thank you for all the support and for being
there fore me.
55
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