Functional aspects of wobble uridine modifications
in yeast tRNA
Anders Esberg
Department of Molecular Biology
Umeå University
Umeå, 2007
Department of Molecular Biology
Umeå University
SE-90187 Umeå, Sweden
Copyright © 2007 Anders Esberg
ISBN: 978-91-7264-302-4
Printed by: Solfjädern Offset, Umeå 2007
2
Table of contents
Abstract………………..…………………...…………………..
5
Main references…..…….………………...……………………
6
1. Introduction
The central dogma.………..……………….....……………..
Transfer RNA……….……..…………………...…………...
The genetic code…….………………...………….…….…..
Translation………….………………………………….……
Translational fidelity….……….…………….………….…..
Translational errors….….…………………….………...…..
Wobble uridine modifications.………………..………….…
The role of wobble uridine modifications in decoding.…….
Proteins required for wobble uridine modifications…..…....
7
8
11
13
17
18
19
21
23
2. Results and discussion…….……………….………………. 27
3. Conclusions………….……………………………………… 42
4. Acknowledgements…………………..……………...……… 43
5. References…………….……………………………….……. 44
6. Papers (I-III)…………….….…………….……….……….. I-III
3
4
Abstract
Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA
into protein. These adaptor molecules require modifications of a subset of their
nucleosides for optimal function. The most frequently modified nucleoside in tRNA
is position 34 (wobble position), and especially uridines present at this position.
Modified nucleosides at the wobble position are important in the decoding process
of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This
thesis addresses the functional aspects of the wobble uridine modifications.
The Saccharomyces cerevisiae Elongator complex consisting of the six
Elp1-Elp6 proteins has been proposed to participate in three distinct cellular
processes; elongation of RNA polymerase II transcription, regulation of polarized
exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we
show that the phenotypes of Elongator deficient cells linking the complex to
ln
transcription and exocytosis are counteracted by increased level of tRNA Gmcm
s UUG
Lys
and tRNA mcm
s UUU . These tRNAs requires the Elongator complex for formation of
the 5-methoxycarbonylmethyl (mcm 5 ) group of their modified wobble nucleoside
5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U). Our results therefore indicate
that the relevant function of the Elongator complex is in formation of modified
nucleosides in tRNAs and the defects observed in exocytosis and transcription are
indirectly caused by inefficient translation of mRNAs encoding gene products
important for these processes.
The lack of defined mutants in eukaryotes has led to limited understanding
about the role of the wobble uridine modifications in this domain of life. In Paper II,
we utilized recently characterized mutants lacking the 2-thio (s 2 ) or
5-carbamoylmethyl (ncm 5 ) and mcm 5 groups to address the in vivo function of
eukaryotic wobble uridine modifications. We show that ncm 5 and mcm 5
side-chains promote reading of G-ending codons, and that presence of a mcm 5 and
an s 2 group cooperatively improves reading of both A- and G-ending codons.
Previous studies revealed that a S. cerevisiae strain deleted for any of the six
Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the
dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit
of zymocin is a tRNA endonuclease that target the anticodon of mcm 5 s 2 U 34
containing tRNAs and that the wobble mcm 5 modification is required for efficient
cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which
are defective in the synthesis of the mcm 5 group.
5 2
5 2
5
Main References:
This thesis is based on the following papers, which are referred to in the text by
their roman numerals.
I
Esberg A., Huang B., Johansson M.J.O., and Byström A.S. (2006).
Elevated levels of two tRNA species bypass the requirement for the
Elongator complex in transcription and exocytosis.
Molecular Cell 24, 139–148.
II Johansson M.J.O., Esberg A., Huang B., Björk G., and Byström A.S.
(2007). Eukaryotic wobble uridine modifications promote a functionally
redundant decoding system.
Manuscript.
III Lu J., Huang B., Esberg A., Johansson M.J.O., and Byström A.S.
(2005). The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
RNA 11, 1648-1654.
6
1. Introduction
The central dogma
The central dogma of molecular biology describes the flow of genetic
information from DNA to protein (Figure 1).
Transcription
DNA
replication
Translation
mRNA
Protein
Figure 1. The central dogma of molecular biology.
DNA is composed of four building blocks: the deoxyribonucleotides
containing the bases Adenine (A), Guanine (G), Cytosine (C), or Thymine
(T). The function of DNA is to provide long-term storage for vital genetic
information, and to function as a blueprint for essential components like
proteins and RNA molecules. The first process in protein synthesis,
transcription, is where an RNA molecule, messenger RNA (mRNA), is
synthesized (transcribed) from a selective DNA region (gene). In the second
process, translation, the mRNA is decoded to generate a specific amino acid
chain (protein) according to the rules defined by the genetic code. The
mRNA nucleotides are decoded in a triplet manner (codons) where each
three-letter code specifies a specific amino acid. However, the mRNA
codons are not directly recognized by amino acids. Translation of mRNA
codons into specific amino acids is dependent on adaptor molecules, which
7
are able to bind a defined amino acid and recognize a specific codon. These
adaptor molecules are small RNA molecules called transfer RNA (tRNA)
that contain a nucleotide-triplet (anticodon) designed to match and decode
the different mRNA codons. The decoding occurs in the ribosome, which is
a large ribonucleoprotein complex capable of binding the mRNA and guide
the tRNA into the correct position on the mRNA. When matching occurs
between codon and anticodon, the amino acid linked to the tRNA will be
incorporated into the growing amino acid chain. This thesis will focus on the
functional aspects of modified uridines present at the first position in the
anticodon.
Transfer RNA
Transfer RNA was discovered by Hoagland et al. 1956 and later described as
a molecule important in translating mRNA into proteins (Hoagland et al.,
1956; For review, RajBhandary and Kohrer, 2006). These small RNA
molecules are composed of four different nucleosides Uridine (U),
Adenosine (A), Guanosine (G), and Cytidine (C). Transfer RNAs are
between 75 and 95 nucleotides in length and usually visualized in the
canonical cloverleaf structure in which the tRNA is divided into the variable
arm, acceptor stem, D-, TΨC-, and anticodon-loop (Figure 2A). All tRNAs
fold into a similar L-shaped three-dimensional structure (Figure 2B)
promoted by base pairs between well-conserved positions within the tRNA
(Dirheimer et al., 1995; Holley, 1965; Holley et al., 1965).
8
In eukaryotes, the RNA polymerase III (Pol III) transcription machinery
transcribes the genes coding for tRNAs. The promoter present in tRNA
genes has internal A- and B-boxes, which correspond to the D- and
TΨC-stem loops of the transcribed tRNA. Transcription by Pol III involves a
multi-step assembly of initiation factors into a pre-initiation complex that
directs recruitment of Pol III. To activate transcription of tRNA genes, the
TFIIIC binds to the A- and B-boxes and acts as an assembly factor to direct
binding of the TFIIIB to an upstream position of the tRNA gene. Assembled
TFIIIB recruits the Pol III enzyme and directs multiple-rounds of
transcription (For review, Schramm and Hernandez, 2002).
A
B
3´
3´
A
C
CCA-end
5´
C
5´
Acceptor-stem
CCA-end
TΨC-loop
D-loop
Variable arm
36
34 35 36
35
34
Anticodon-loop
Anticodon-loop
Figure 2. Schematic structure of a tRNA. (A) Two-dimensional structure. (B)
Tertiary-structure (Kim et al., 1974). Positions 34, 35, and 36 (anticodon), are shown
in black.
9
All eukaryotic tRNA molecules are transcribed as precursors with extra
sequences at both the 5´ and 3´ ends, which have to be processed to generate
a mature tRNA. Removal of the 5´ leader is accomplished by a
ribonucleoprotein, RNaseP, whereas the 3´ end is processed by the
endoribonuclease RNaseZ. The primary tRNA transcript from eukaryotic
cells does not contain the 3´ CCA sequence, which is essential for
aminoacylation (Figure 2). The 3´ CCA sequence is added by the
tRNA-nucleotidyltransferase. Ten S. cerevisiae tRNA species contain an
intervening sequence in their primary transcript. The introns can vary in size
between 14-60 nucleotides, and are always located one nucleotide 3´ of the
anticodon. Splicing involves multiple-steps, first, the intron is removed by
the tRNA-splicing endonuclease. Secondly, the RNA-ends are ligated
together by the tRNA-ligase, and finally the 2´ phosphate group present after
ligation is removed by a phosphotransferase. For the fidelity of translation,
each tRNA must be charged with its cognate amino acid. The
aminoacyl-tRNA synthetases (aaRS) link the correct amino acid to the 3´
CCA end of the cognate tRNA. In general, there is one aaRS for each of the
20 amino acids used in protein synthesis. Finally, to generate a fully
functional adaptor molecule, specific tRNA modifications have to be
introduced post-transcriptionally. The different modified nucleosides present
in the tRNA are derivatives of the normally present nucleotides A, C, G, or U,
and up till now twenty-five different tRNA modifications have been found in
cytoplasmic S. cerevisiae tRNA (For reviews, Björk, 1995; Hopper and
Phizicky, 2003; Ibba and Söll, 2000; Johansson and Byström, 2005).
10
The genetic code
The mRNA sequence is decoded in a triplet manner into a specific amino
acid sequence by a three-nucleotide complementary region, the anticodon, in
the tRNA (Figure 3).
3´ 5´
36 35 34
U U U
5´
XXX
AI AII AIII
mRNA
ZZZ
3´
Figure 3. A schematic drawing of codon-anticodon interaction. The mRNA codon is
composed of three nucleotides A I , A II , and A III . The anticodon of the tRNA is
composed of three nucleotides at position 34, 35, and 36. U 36 will interact with the
first position (A I ), U 35 with the second- (A II ), and U 34 (wobble base) with the
third-position (A III ) of the mRNA codon.
The deciphering of the three-letter codons generated what we today call the
“Genetic Code”. The genetic code is in principle universal, consisting of 64
triplet letters, where 61 codons code for amino acids and three codons
terminate translation . The genetic code is degenerate, i.e., many amino acids
are specified by more than one codon. Two observations provide an
explanation for the degeneracy of the genetic code. First, the existence of
11
isoaccepting tRNA species charged with the same amino acid. Second, the
“wobble hypothesis” proposed by Crick, which predicts that some tRNA
species can decode more than one codon (Crick, 1966; Crick et al., 1961).
The wobble hypothesis stated that the nucleoside at the first position of the
anticodon (wobble position) could form either a canonical or a wobble base
pair with the third nucleoside in the codon, provided that the nucleosides at
position 35 and 36 in the tRNA form Watson-Crick base pairs with the first
two nucleosides of the codon (Crick, 1966) (Figure 3).
The “Codon Table” summarizes all the 64 possible triplets and clarifies
which codon specifies which amino acid (Figure 4). The two first letters of
the three letter codon create 16 different combinations, constructing the 16
codon boxes in the codon table (Figure 4). There are two types of codon
boxes, the family- and the split-codon boxes. In a family codon box, all four
codons code for the same amino acid, whereas a split codon box contains
codons for more than one amino acid.
12
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Phe
Leu
Leu
Ile
Met
Val
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Pro
Thr
Ala
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
Tyr
Stop
His
Gln
Asn
Lys
Asp
Glu
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
Cys
Stop
Trp
Arg
Ser
Arg
Gly
Figure 4. The codon table. The genetic code is composed of four different letters U, C,
A, and G, generating in total 64 different three-letter combinations. The codon table
summarizes all 64 codons and clarifies which codon specifies which amino acid.
Translation
Protein translation is one of the most conserved processes in gene expression.
Translating mRNA into protein requires three different RNA species, mRNA,
tRNA, and rRNA. In addition, this process also requires a number of
proteins important for assistance throughout the translation process. The
eukaryotic ribosome is a large multi-subunit complex composed of 4 RNA
molecules and ~76 proteins, which assemble into the small 40S and the large
60S subunit, which together form the mature 80S ribosome. The ribosome is
13
capable of binding tRNA in three separate binding sites: the A-site, P-site,
and E-site. Each of these sites has a distinct function, the A-site binds the
a minoacyl-tRNA, the P-site binds the p eptidyl-tRNA, and the E-site
( E xit-site) binds the deacyl-tRNA. Translation of mRNA into protein can be
divided into three distinct steps, initiation, elongation, and termination.
Initiation of translation is a sequence of events important for
identification of the correct start codon and determination of the correct
reading frame. According to the scanning model of translation initiation, the
pre-initiation complex (40S, initiation factors, and the initiator methionine
Met
tRNA CAU
) binds to the 5´ end of the mRNA (Figure 5A). The pre-initiation
complex scans the mRNA in the 5´ to 3´ direction searching for the start
codon (Figure 5B). Once the start codon is identified, the initiation factors
are released and the 60S is recruited, assembling the 80S ribosome (Figure
5C). There are three potential reading frames in an mRNA and the reading
frame is fixed by the identification of the start codon. Normally a reading
frame is maintained and the mRNA translated in one continuous motion
from start to finish (Hershey and Merrick, 2000).
During translation elongation, the ribosome moves stepwise on the
mRNA, codon by codon, guiding the tRNA into the correct position. The
ribosome faithfully selects aminoacyl-tRNA based on how well it fits into
the A-site and the strength of the codon-anticodon interaction. Each
elongation cycle will add one amino acid to a growing amino acid chain. An
elongation cycle starts when a ternary complex, consisting of the
aminoacyl-tRNA, elongation factor 1A (eEF1A), and GTP, enters the
14
ribosomal
A-site,
(Figure
5D).
If
matching
occurs
between
the
codon-anticodon, hydrolysis of the GTP promotes eEF1A-GDP release
(Figure 5E). The amino acid chain is transferred from the P-site tRNA to the
A-site tRNA by the enzymatic activity of the ribosome peptidyl transferase
center (Figure 5F). In this process, a new amino acid is added to the growing
amino acid chain. Following peptide transfer, the elongation factor 2 (eEF2)
promotes translocation of the peptidyl-tRNA to the P-site (Figure 5G). In
addition to eEF1A and eEF2, elongation factor 3 (eEF3) promotes release of
the E-site tRNA from the ribosome, and the elongation factor 1B (eEF1B)
recycles the eEF1A-GDP to eEF1A-GTP (Figure 5) (Merrick and Nyborg,
2000).
Of the 64 different codons, three are designated to terminate protein
translation (UAA, UAG, and UGA) (Figure 4). These codons are not
decoded by a tRNA into an amino acid, instead these codons are recognized
by the eukaryotic release factor 1 (eRF1) (Figure 5H). When the eRF1 binds
a stop codon, it will together with release factor 3 (eRF3) promote
translation termination. In this process, the amino acid chain dissociates
from the P-site tRNA, the mRNA is released, and ribosomal subunits
dissociate (Figure 5I) (Welch et al., 2000).
15
Initiation
Termination
eRF3
eRF1
A
(AUG)
5´Cap
E
P
H
A
I
eRF1
40S
eRF3
B
(STOP)
E
5´Cap
E
P
P
A
A
Elongation
C
60S
E
P
A
40S
Proofreading
step 1
eEF3
G
D
eE
F1
A
GTP
E
P
E
A
A
GDP
eE
F1
eEF2
F
Peptidyl
transfer
P
A
eEF1B
Translocation
E
P
A
E
Proofreading
step 2
E
P
A
Figure 5. Simple view of eukaryotic protein translation. (A-B) Initiation of translation.
(C-G) Translation elongation. (H-I) Translation termination.
16
Translational fidelity
A vital task in protein synthesis is to incorporate the correct amino acid and
maintain the correct reading frame at the same time sustaining speed. On
average, the translation machinery incorporates ~20 amino acids per second;
however, the machinery is not perfect since an incorrect amino acid is
occasionally incorporated. Cells have in the course of evolution, evolved
numerous proofreading steps to improve translational accuracy.
To assure translational fidelity, each tRNA species has to be charged
with its cognate amino acid. To increase the accuracy of tRNA
aminoacylation, the aaRS accepts only the correct amino acid into the
substrate-binding site. To further reduce misaminacylation of incorrect
substrate tRNAs, a number of identity elements are present within the
different tRNA species. In general, the identity element lies in the two distal
parts of the tRNA, with the major determinants in the anticodon loop and the
acceptor stem. Moreover, modified nucleosides, especially those located in
the anticodon region of the tRNA, have been shown to be important for
charging (For reviews, Giege et al., 1998; Ibba and Söll, 2000).
According to current models, two independent proofreading steps assure
selection of a correct aminoacyl-tRNA into the A-site of the ribosome
(Thompson, 1988). The first proofreading step involves the initial binding of
the ternary complex to the ribosome. Here the primary strength of the
codon-anticodon interaction determines if the tRNA should be accepted into
the ribosome (Figure 5D). The second proofreading step is after GTP
hydrolysis and release of the eEF1A-GDP, here the ribosome senses the
17
codon-anticodon strength and the A-site fitness, and together these features
determine whether the aminoacyl-tRNA should be rejected or not (Figure 5E)
(For review, Ogle and Ramakrishnan, 2005). In the selection of the cognate
aminoacyl-tRNA into the A-site of the ribosome, modifications present at
the wobble position have been shown to play a vital role, primarily by
influencing the codon-anticodon interaction (Agris, 1991; Lim, 1994; Takai
and Yokoyama, 2003; Yokoyama and Nishimura, 1995; Yokoyama et al.,
1985). In addition to these proofreading steps, the E-site tRNA has also been
shown to be important for the selection of correct A-site tRNA, but the
mechanism for this proofreading step is not clear (Nierhaus, 2006).
Translational errors
Even though the translational machinery is capable of translating long
mRNA faithfully, errors now and then occur. During the translation
elongation process, three classes of translational errors can be distinguished.
(I) Progressive errors, the release of either a shorter or a longer form of
the amino acid chain. At least three different events can cause this kind of
translational error, (1) read through of stop codons, (2) incorrect
translational termination, or (3) drop-off of peptidyl-tRNA.
(II) Missense errors, an incorrect amino acid incorporated at sense
codons. This kind of error is usually caused by misreading of the third
nucleotide of the mRNA codon by the wobble base of the tRNA. In addition,
tRNA can also be misaminoacylated and thereby incorporate an incorrect
amino acid even though it pairs with the matching codon.
18
(III) Frameshifting errors, a shift in the reading frame. Hypothetically,
there are four different ways to induce frameshifting: (1) Transcriptional
errors
expanding
or
contracting
the
codon
size,
(2)
incoming
aminoacyl-tRNA binds out of frame, (3) translocation error, or (4) slippage
of the peptidyl-tRNA. After a frameshifting error, the original genetic
message is completely altered and can produce either longer or shorter form
of proteins.
It is broadly accepted that a single mRNA that encodes for a defined
amino acid chain will give rise to identical protein products, assuming that
these proteins go through the same post-translational modifications. This
notion was recently challenged by the findings that altered translational rate
could
change
protein
folding
and
modify
the
protein
function
(Kimchi-Sarfaty et al., 2007). It was hypothesized that temporal separation
of folding events throughout synthesis of a protein molecule might be vital
to ensure correct protein function. This implies that cells have a high
demand for a fully functionally translational machinery to maintain correct
translational speed to assure accurate protein folding.
Wobble uridine modifications
Transfer RNA modifications are found in all organisms and all over the
tRNA molecule. These modifications are introduced post-transcriptionally
and are derivatives of the normal nucleotides A, C, U, and G (For reviews,
Björk, 1995; Johansson and Byström, 2005). So far, 91 different tRNA
modifications have been found in the three kingdoms of life. Some tRNA
19
modifications are present at the same position, in the same population of
tRNA, and within all phylogenic domains, suggesting a conserved function
(Björk et al., 2001; [email protected]).
The modifications present in tRNA have been shown to play a vital role
in numerous processes e.g., folding and stability of tRNA (Anderson et al.,
1998; Helm, 2006), recognition and anti-determinants for proteins
interacting with tRNAs (Åström and Byström, 1994; For review, Giege et al.,
1998), and intracellular localization of tRNA (Kaneko et al., 2003). The
most frequently modified base in tRNA is positions 34, and especially tRNA
carrying a uridine at the wobble position. Modifications at wobble position
are primarily thought to influence the decoding properties of the tRNA.
In total, there are 42 cytoplasmic S. cerevisiae tRNA species, and out of
these, thirteen contain a uridine at the wobble position in the primary
transcript. Of the thirteen U 34 containing tRNA species, one carries an
unmodified U ( tRNA Leu
) and one has a pseudouridine (Ψ) ( tRNA ΨIleAΨ ). The
UAG
remaining eleven U 34 containing tRNAs have either a 5-carbamoylmethyl
(ncm 5 ) or a 5-methoxycarbonylmethyl (mcm 5 ) group at the position 5
(Figure 6) (in many cases the ncm 5 and the mcm 5 modifications will be
referred to as xm 5 ) (Huang et al., 2005; For review, Johansson and Byström,
2005) (Paper II). Three of the mcm 5 containing tRNAs are thiolated at
position
2,
generating
the
hyper
modified
5-methoxycarbonylmethyl-2-thiouridine nucleoside (mcm 5 s 2 U) (Kobayashi
et al., 1974; Smith et al., 1973) (Paper III) (Figure 6).
20
O
O
HN
HN
N
O
HO
O
HO
O
O
O
CH 2 CNH 2
HN
O
N
HO
O
O
O
CH 2COCH 3
N
O
HN
S
HO
O
CH 2COCH 3
N
O
OH OH
OH OH
OH OH
OH OH
U
ncm5U
mcm5U
mcm5s2U
Figure 6. Examples of uridine or uridine derivatives present at position 34 (wobble
position).
From
the
left;
uridine
(U),
5-carbamoylmethyl-uridine
(ncm 5 U),
5
5-methoxycarbonylmethyl-uridine (mcm U), and 5-methoxycarbonylmethyl-2-thio
-uridine (mcm 5 s 2 U).
The role of wobble uridine modifications in decoding
In the decoding process, an aminoacyl-tRNA can potentially direct decoding
of three different types of codons. Cognate codon: the tRNA is able to form
three Watson-Crick base pairs, Near-cognate codons: two Watson-Crick base
pairs and one wobble base pair at the third position, and Non-cognate codon:
one or less Watson-Crick base pair. In the translational process, a
unmodified U 34 will according to Crick’s original wobble hypothesis
preferentially decode cognate A- and near cognate G-ending codons,
assuming that the nucleotides at position 35 and 36 in the tRNA form
Watson-Crick base pairs with the two first nucleotides of the mRNA codon
(Crick, 1966). At the time when Crick presented his hypothesis, it was not
known that cytoplasmic tRNA almost never contains an unmodified U 34 .
Since then, Crick’s hypothesis has been revised and it was suggested that an
21
unmodified U 34 recognizes all four nucleotides U, C, A, and G, whereas a
modified uridine can alter the decoding capacity (Agris, 1991; Björk, 1995;
Lim, 1994; Takai and Yokoyama, 2003; Yokoyama and Nishimura, 1995;
Yokoyama et al., 1985). Based on results from in vitro studies, as well as
structural considerations, it has been proposed that the presence of an xm 5
group at a wobble uridine would prevent pairing with U- and C-ending
codons (Lim, 1994; Yokoyama and Nishimura, 1995; Yokoyama et al., 1985).
For the decoding of A- and G-ending codons, two models are proposed. The
first and most accepted model states that presence of an xm 5 side-chain at
U 34 improves reading of A- and causes inefficient decoding of G-ending
codons (Yokoyama and Nishimura, 1995; Yokoyama et al., 1985). The
second model suggests that an xm 5 U nucleoside efficiently reads both Aand G-ending codons (Kruger et al., 1998; Lim, 1994; Murphy et al., 2004;
Takai and Yokoyama, 2003; Yarian et al., 2002).
Three mcm 5 U 34 containing tRNAs are thiolated at position 2 of the
base. Interestingly, the 2-thio group is only found in tRNAs decoding in split
codon boxes. Similar to an xm 5 U 34 nucleoside, presence of an mcm 5 s 2 U 34
residue was suggested to prevent pairing with U- and C-ending codons,
allow efficient reading of A-, and simultaneously reduce the ability to pair
with G-ending codon (Lustig et al., 1981; Sekiya et al., 1969; Yokoyama et
al., 1985). More recent data have indicated that a mcm 5 s 2 U 34 residue would
allow reading of both A- and G-ending codons (Murphy et al., 2004).
22
Proteins required for wobble uridine modifications
Today over 50 gene products are dedicated for synthesis of tRNA
modifications in S. cerevisiae (Huang et al., 2005; For review, Johansson
and Byström, 2005; Nakai et al., 2007) (Lu J. personal communication).
Surprisingly, at least twenty-four of these are required for the synthesis of
wobble uridine modifications.
At this moment, fourteen gene products have been identified that are
required for synthesis of the xm 5 modification at U 34 . The Elongator
complex, consisting of six proteins Elp1-Elp6, is required for the formation
of ncm 5 and mcm 5 side-chains at wobble uridines (Figure 6) (Huang et al.,
2005). In addition to a role in tRNA modification, the Elongator complex
was proposed to participate in two other distinct cellular processes.
Originally the Elongator complex was identified based on its association
with the hyper-phosphorylated elongating form of RNA polymerase II (Pol
II) (Otero et al., 1999). Purified Elongator complex from yeast cells is able
to transfer acetyl groups from acetyl-CoA to histones H3 and H4 in vitro
(Hawkes et al., 2002; Kim et al., 2002; Winkler et al., 2002; Wittschieben et
al., 1999). Moreover, an elp3Δ strain shows a decreased acetylation level of
primarily histone H3 in vivo (Winkler et al., 2002). Together these data
suggested that the Elongator complex mediates Pol II transcription
elongation through chromatin structure. Another distinct function described
for the Elongator complex is in polarized transport of secretory vesicles to
the bud tip (Rahl et al., 2005). This transport event requires activation of the
vesicle-associated GTPase Sec4p by its guanine nucleotide exchange factor
23
Sec2p (Salminen and Novick, 1987; Walch-Solimena et al., 1997). It was
found that Sec2p associated with Elp1p and that polarized localization of
Sec2p was dependent on the presence of Elp1p, suggesting that the
Elongator complex regulates exocytosis by influencing the localization of
Sec2p (Rahl et al., 2005). In this thesis, we address whether the Elongator
complex has three distinct functions or whether it regulates one key process
that sequentially leads to downstream effects in the two other processes
(Paper I). We also utilized an elp3 null mutant to address the function of the
ncm 5 and mcm 5 side-chains at wobble uridines (Paper II). In addition to the
elp1–elp6 mutants, the formation of ncm 5 and mcm 5 modified wobble
uridines is abolished in the kti11, kti12, sit4, kti14, and the double sap185
sap190 mutants, whereas a kti13 mutant show significantly reduced levels
(Huang et al., 2005) (Lu J. personal communication).
The esterified methyl constituent of the mcm 5 group is synthesized by
the tRNA carboxyl methyltransferase, Trm9 protein (Kalhor and Clarke,
2003). The TRM9 gene was identified in a search for S. cerevisiae proteins
that contained putative AdoMet binding motifs, and later it was shown that
Trm9p used AdoMet as the donor for the tRNA methylation reaction (Kalhor
and Clarke, 2003; Niewmierzycka and Clarke, 1999). A strain deleted for the
TRM9 gene did not contain mcm 5 U or mcm 5 s 2 U in total tRNA (Figure 6),
and lacked methyl-esterified nucleosides in purified tRNA Arg and tRNA Glu .
Extracts from wild-type but not the trm9 mutant catalyzed incorporation of
methyl-esters into a substrate tRNA. This suggested that the Trm9 protein
catalyzed formation of methyl-esters in both mcm 5 U and mcm 5 s 2 U in
24
tRNA (Kalhor and Clarke, 2003). In this thesis, we used a trm9 null mutant
to address the role of the esterified methyl group in decoding (Paper II).
Glu
tRNA Lys
Three tRNAs, tRNA Gln
mcm 5s 2 UUG ,
mcm5s 2 UUU , and tRNA mcm 5s 2 UUC carry
the hyper-modified nucleoside mcm 5 s 2 U at the wobble position (Figure 6).
At the moment, there are ten gene products known to be required for the
synthesis of the 2-thio group at U 34 . The Tuc1 protein was identified by
sequence homology to the Escherichia coli TtcA protein, which is required
for the synthesis of s 2 C in position 32 of a subset of tRNAs (Björk G.
personal communication). However, no s 2 C modification has been identified
in S. cerevisiae tRNA, so it was speculated if this protein could be required
for the formation of the 2-thio group present in the mcm 5 s 2 U 34 nucleoside.
Accordingly, total tRNA isolated from a tuc1 null strain lack the s 2 group of
the mcm 5 s 2 U wobble nucleoside (Björk G. personal communication). The
Kluyveromyces lactis zymocin is a heterotrimeric toxin consisting of a α, β,
and γ subunit (Schaffrath and Meinhardt, 2005). The γ-toxin subunit is a
tRNA
endonuclease
cleaving
,
tRNA Gln
mcm 5s 2 UUG
tRNA Lys
,
mcm5s 2 UUU
and
5 2
tRNA Glu
mcm 5s 2 UUC at the 3’ side of the wobble nucleoside mcm s U (Paper III).
Presence of the mcm 5 s 2 U 34 nucleoside is important for efficient cleavage of
substrate tRNA and mutants defective in formation of either mcm 5 or s 2
side-chains are resistant to zymocin (Paper III) (Lu J. personal
communication). In a screen for zymocin resistant mutants, we identified
five mutants defective in formation of the s 2 group of the mcm 5 s 2 U 34 ,
among these the tuc1 and the tuc2 mutants (Lu J. personal communication).
In addition to Tuc1p and Tuc2p, the Nfs1p, Yor251Cp, Nbp35p, Cia1p,
25
Urm1p, Uba4p, Isu1p, and Isu2p are also required for the formation of s 2 U
at wobble position (Nakai et al., 2007) (Lu J. personal communication). In
this thesis, we use the tuc1 and tuc2 mutant (also called NCS6 and NCS2,
respectively) to address the function of the 2-thio group in decoding (Paper
II).
26
2. Results and discussion
Paper I: Elevated levels of two tRNA species bypass the
requirement for the Elongator complex
in
transcription
and
exocytosis.
The S. cerevisiae Elongator complex, consisting of the Elp1-Elp6 proteins,
has been proposed to participate in three distinct cellular processes:
transcriptional elongation, exocytosis, and formation of modified wobble
uridines in tRNA. It was important to clarify whether the Elongator complex
has three distinct functions or whether it regulates one key process that
sequentially leads to downstream effects. A strain lacking Elongator function
displays a multitude of phenotypes, including temperature sensitive (Ts)
growth at 38°C (Frohloff et al., 2001; Jablonowski et al., 2001a; Krogan and
Greenblatt, 2001).
We investigated if there were genes in high dosage that could bypass the
requirement for an elp3 null mutant. Except for plasmids carrying the ELP3
gene, we identified five plasmids that partially suppressed the Ts phenotype.
All these plasmids contained inserts carrying a tK(UUU) gene, which codes
Lys
for tRNA mcm
. In a strain lacking Elongator function, this tRNA species
5 s 2 UUU
contains s 2 U rather than mcm 5 s 2 U at the wobble position. Since the
ELP1-ELP6 genes are required for formation of wobble ncm 5 and mcm 5
side-chains in 11 tRNA species (Huang et al., 2005) (Paper II) (Paper III),
we investigated whether elevated levels of any of the remaining 10 tRNAs
27
species would suppress the Ts phenotype of the elp3Δ strain. Weak
suppression of the Ts phenotype of the elp3Δ strain was obtained by
increased dosage of a tQ(UUG) gene which codes for mcm 5 s 2 U containing
tRNA Gln
. Simultaneously increased expression of the tK(UUU) and
mcm 5s 2 UUG
tQ(UUG) genes in an elp3Δ strain resulted in a cooperative growth
improvement at both 30°C and 38°C. The tRNA Gln
species contain the
mcm 5s 2 UUG
same wobble nucleoside (mcm 5 s 2 U) as tRNA Lys
and contain s 2 U in
mcm 5s 2 UUU
Elongator deficient cells (Paper III). Because strains with a mutation in any
of the ELP1-ELP6 genes display similar phenotypes, we tested if the
suppression generated by increased dosage of the tK(UUU) and tQ(UUG)
genes is also true in other Elongator mutants. Increased dosage of the
tK(UUU) and tQ(UUG) genes suppressed the Ts phenotype of all the
elp1-elp6 mutants. These results indicate with respect to growth defects,
elevated levels of the hypomodified tRNA sGln
and tRNA sLys
species
2 UUG
2 UUU
bypass the requirement for the Elongator complex.
The second function proposed for the Elongator complex was
acetylation of histones, and the histone acetyl transferase (HAT) activity was
primarily associated with the Elp3 protein. HATs transfer acetyl groups from
acetyl-CoA to the amino group of certain lysine residues within histone
N-terminal tails (Sterner and Berger, 2000). Lysine 14 in histone H3 has
been shown to be the major target for the HAT activity of Elp3p (Kuo et al.,
1996; Winkler et al., 2002). Histones isolated from elp3Δ cells with or
without elevated levels of tRNA sLys
and tRNA sGln
were analyzed with
2 UUU
2 UUG
anti-histone H3 and anti-acetyl-lys14-histone H3 antiserum. Increased levels
28
of the hypomodified tRNA sLys
and tRNA sGln
restored the Lysine 14
2 UUU
2 UUG
acetylation levels of histone H3 in elp3Δ cells. Thus, the low acetylation
level of Lysine 14 in histone H3 isolated from elp3Δ cells is most likely
caused by a reduced function of the hypomodified tRNA sLys
and
2 UUU
tRNA sGln
, strongly questioning a role of Elp3p as a bona fide HAT.
2 UUG
The third distinct function proposed for the Elongator complex was the
delivery of transport vesicles to the bud-site (Rahl et al., 2005). Sec2p is a
guanine nucleotide exchange factor that triggers GDP to GTP exchange of
Sec4p (Walch-Solimena et al., 1997). Activated GTP-bound Sec4p
participates in delivery of vesicles to a specific region of the plasma
membrane (Salminen and Novick, 1987) (Walch-Solimena et al., 1997). A
sec2-59 mutant displays a Ts phenotype (Nair et al., 1990) and introduction
of an elp1 null allele into the sec2-59 mutant strain suppress the Ts
phenotype (Rahl et al., 2005). Increased expression of the hypomodified
and tRNA sGln
in the elp1Δ sec2-59 double mutant generated a
tRNA sLys
2 UUU
2 UUG
Ts phenotype, as observed in the sec2-59 single mutant. Thus, increased
levels of these tRNAs mimic the presence of Elp1p. Sec2p is a cytosolic
protein that normally concentrates in a distinct polarized manner to the site
of exocytosis (Elkind et al., 2000). In an elp1Δ strain, Sec2p is mislocalized
and it was suggested that a physical interaction between Elp1p and Sec2p
was important for the polarized localization of Sec2p (Rahl et al., 2005).
Elevated levels of the hypomodified tRNA sLys
and tRNA sGln
in the
2 UUU
2 UUG
elp1Δ strain restored polarized localization of Sec2p. This shows that an
interaction to Elp1p is not a requirement for a polarized localization of
29
Sec2p, rather it reflects the importance of the mcm 5 side-chain in
and tRNA Gln
for post-transcriptional expression of
tRNA Lys
mcm 5 s 2 UUU
mcm 5s 2 UUG
gene products crucial in the exocytosis process.
The mcm 5 or s 2 groups of the wobble mcm 5 s 2 U residue are both
believed to affect decoding in a similar manner (see above). Phenotypes
caused by removal of the s 2 or the mcm 5 group in tRNA Lys
and
mcm 5 s 2 UUU
tRNA Gln
are expected to be a consequence of less efficient decoding
mcm 5s 2 UUG
Lys and Gln codons. We recently found that a strain with a tuc2 (also called
ncs2) null allele lacks the s 2 group in mcm 5 s 2 U containing tRNA species
(see above). A tuc2 strain displays similar phenotypes as elp1 and elp3
mutant
strains
e.g.,
delayed
transcriptional
activation,
chromatin
remodeling-, and exocytosis-defects. Increased levels of the mcm 5
Lys
containing tRNA mcm
and tRNA Gln
suppressed the phenotypes of a
5 UUU
mcm 5 UUG
tuc2 null strain, suggesting that the defects were caused by lack of the 2-thio
group at wobble position. We conclude that phenotypes proposed to be
caused by a direct effect of Elongator on exocytosis or transcription are
observed in a tuc2 mutant where the functionality of tRNA Lys
and
mcm 5 s 2 UUU
has been reduced by an Elongator-independent mechanism.
tRNA Gln
mcm 5s 2 UUG
Summary:
In Paper I, we show that increased levels of the hypomodified tRNA sLys
2 UUU
and tRNA sGln
could bypass the requirement for the Elongator complex in
2 UUG
transcription and exocytosis. As the Elongator complex is required for the
formation of the mcm 5 group at position 34 in these tRNAs, we suggest that
30
the relevant function of the Elongator complex is to modify wobble uridine
in tRNA, and that transcription and exocytosis defects in Elongator mutant
are indirect effects resulting from a primary defect in translation (Figure 7).
Elongator
complex
Pol II
m
R
N
A
Sec2
mcm5s2
Exocytosis
tRNA modification
Transcription
Figure 7. Model: The Elongator complex is required to modify uridines at the wobble
position. The defects observed in exocytosis and transcription are indirectly caused
by inefficient translation of mRNAs encoding gene products important for these
processes.
31
Paper II: Eukaryotic wobble uridine modifications promote a
functionally redundant decoding system
Modified nucleosides in the anticodon region of tRNA are important in the
decoding process. Since defined mutants defective in formation of the s 2 or
ncm 5 and mcm 5 groups at wobble uridines have not been available, the in
vivo roles of these modifications have not been investigated. The Elongator
complex was recently shown to be required for formation of ncm 5 and mcm 5
side-chains at position 34 (Huang et al., 2005). In addition, the last methyl
group in the mcm 5 side-chain is synthesized by the Trm9p (Kalhor and
Clarke, 2003). Furthermore, the Tuc2p is required for the formation of the
2-thio group in mcm 5 s 2 U 34 containing tRNA species (Paper I) (Björk G.
personal communication). Strains with mutations in any of the ELP1-ELP6,
TRM9, or TUC2 genes are viable, making it possible to investigate the in vivo
role of the wobble ncm 5 , mcm 5 , and s 2 groups.
According to current models based on mainly physicochemical studies, a
tRNA with an unmodified U 34 may decode codons ending with any of the
four nucleotides (Lim, 1994; Yokoyama and Nishimura, 1995). To
investigate if an unmodified wobble uridine is able to read codons ending
with any of the nucleotides in vivo, we utilized the distribution of tRNA
species in the Leucine and Proline family codon boxes. In the Leucine box,
there are only two tRNA species present, the tRNA Leu
containing an
UAG
Leu
unmodified U 34 (Randerath et al., 1979), and the tRNA GAG
harboring a G 34 .
Leu
Interestingly, a strain lacking the G 34 containing tRNA GAG
species is viable,
32
suggesting that an unmodified wobble uridine can read all four nucleotides.
In the Proline box, there are two tRNA species present, one contains
ncm 5 U 34 ( tRNA Prncmo 5 UGG ) and the other one contains an A at the wobble
position (most likely converted to I 34 ). A strain lacking the A 34 containing
o
tRNA PrAGG
species is viable with no apparent growth defect. Introduction of
an elp3Δ allele into this strain did not generate a synthetic growth defect,
o
is also able to decode
suggesting that the unmodified U 34 in tRNA PrUGG
codons ending with any of the four nucleotides. The ability of tRNA Prncmo 5 UGG
to decode U- and C-ending codons is independent of the ncm 5 group, which
could imply that reading of these codons involves a two out of three
interaction (Lagerkvist, 1978).
To investigate if the ncm 5 group in tRNA Val
is important for
ncm 5 UAC
decoding A-ending codons, we reduced the copy number of the genes coding
in yeast cells lacking the elp3 allele. In a
for ncm 5 containing tRNAVal
ncm5 UAC
strain deleted for one of the two genes coding for tRNA Val
, introduction
ncm 5 UAC
of the elp3Δ allele did not generate a synthetic growth defect. Since a tRNA
species with an I 34 residue is present in the Valine family codon box
( tRNA Val
), it is possible that lack of a synergistic phenotype is due to the
IAC
ability of the I 34 containing tRNA to decode GUA codons. However, a strain
species is inviable, showing that
lacking the ncm 5 containing tRNA Val
ncm5 UAC
the I 34 containing tRNA Val
cannot decode the GUA codons. We conclude
IAC
that the absence of an ncm 5 side-chain at U 34 does not reduce the ability of
the tRNA Val
to decode the GUA codons.
ncm 5 UAC
is important for
To investigate if the ncm 5 group in tRNA Val
ncm 5 UAC
33
decoding G-ending codons, we deleted the two genes coding for the C 34
Val
containing tRNA CAC
species. As this strain was viable, the ncm 5 U 34
containing tRNA Val
must be able to decode GUG codons. However,
ncm 5 UAC
introduction of the elp3 allele into a strain lacking the C 34 containing
Val
tRNA CAC
generated lethality, showing that the ncm 5 group in tRNA Val
ncm 5 UAC
is required for reading the GUG codons. We obtained similar results using
comparable genetic strategies for the Threonine and the Serine codon boxes.
Together these data supports a model where the ncm 5 U 34 nucleoside does
not enhance the ability to decode A-, but improves reading of G-ending
codons.
To test if the mcm 5 group at U 34 is important for decoding A-ending
codons, we deleted two of the three genes coding for the mcm 5 U 34
containing tRNA Gly
. Introduction of the elp3Δ allele into this strain did
mcm 5 UCC
not generate a synthetic growth defect, suggesting that the mcm 5 group does
not notably affect the ability of tRNA Gly
to decode GGA codons. To
mcm 5 UCC
address the role of the mcm 5 group in decoding G-ending codons, we
introduced an elp3Δ allele into a strain lacking the C 34 containing tRNA Gly
CCC
species. A synthetic growth defect was observed, suggesting that the
presence of a mcm 5 side-chain improves decoding of G-ending codons. We
obtained similar results using a comparable genetic approach in the Arginine
split codon box (AGN) to address the role of the mcm 5 group in decoding
G-ending codons. Taken together, this suggests that the mcm 5 residue
improves reading of G-, but has no significant influence on decoding
A-ending codons.
34
To investigate the role of the mcm 5 s 2 U 34 residue in the decoding of
G-ending codons, a strain with a deletion of the single copy C 34 containing
ln
tRNA GCUG
gene was constructed. This strain is inviable, suggesting that
ln
is unable to read the G-ending codons. The presence of either
tRNA Gmcm
5s 2 UUG
a mcm 5 or an s 2 group could potentially prevent reading of G-ending
codons. To test this hypothesis we deleted the single copy C 34 containing
ln
tRNA GCUG
gene in either an elp3Δ or a tuc1Δ null strain. Lack of neither the
mcm 5 nor the s 2 group suppressed the inviability of a strain lacking the C 34
ln
ln
containing tRNA GCUG
species. Thus, normal levels of the tRNA Gmcm
5s 2 UUG
carrying either the mcm 5 s 2 U 34 , mcm 5 U 34 or the s 2 U 34 at wobble position
are not able to decode G-ending codons. Interestingly, elevated levels of the
ln
ln
tRNA Gmcm
suppressed the need for C 34 containing tRNA GCUG
in a
5s 2 UUG
wild-type background. This suppression was not observed in an elp3Δ or
tuc1Δ background, suggesting that the mcm 5
and the s 2
groups
cooperatively improve decoding of G-ending codons.
Summary
According to current models, an unmodified U 34 should be able to decode all
four nucleotides U, C, A, and G (Lim, 1994; Yokoyama and Nishimura,
1995). This is true for two of the tRNA species we have investigated. We
o
show that the tRNA Leu
and the hypomodified tRNA PrUGG
, both containing
UAG
an unmodified U 34 , are able to decode codons ending with any of the four
nucleosides. However, the decoding ability of U 34 is highly specific as an
unmodified U 34 in tRNA Thr
or tRNA Ser
, is incapable of reading their
UGU
UGA
35
respective G-ending codons. Therefore, we suggest that it is rather an
exception than a role for an unmodified U 34 to be able to decode all four
nucleotides and we do not exclude the possibility that reading of these
codons could involve a two out of three interaction (Lagerkvist, 1978).
According to current models, presence of an xm 5 side-chain is
predicted to either restrict reading to A- or allow efficient decoding of both
A- and G-ending codons (Kruger et al., 1998; Lim, 1994; Murphy et al.,
2004; Takai and Yokoyama, 2003; Yarian et al., 2002; Yokoyama and
Nishimura, 1995; Yokoyama et al., 1985). We found no evidence for a role
of the xm 5 side-chain in tRNA Val
, tRNA Ser
, and tRNA Gly
in
ncm 5 UAC
ncm 5 UGA
mcm 5 UCC
the decoding of their respective A-ending codons. However, we could show
that presence of an xm 5 side-chain in tRNA Val
ncm 5 UAC
tRNA Ser
,
ncm 5 UGA
, and tRNA Gly
improves reading of G-ending codons.
tRNA Arg
mcm 5 UCC
mcm 5 UCU
The presence of a mcm 5 s 2 U 34 residue was originally proposed to allow
the tRNA to efficiently read the cognate A-ending codon and simultaneously
reduce the ability to decode G-ending codons (Lustig et al., 1981; Sekiya et
al., 1969; Yokoyama et al., 1985). Together the data presented in Paper I and
Paper II, suggests that the presence of the mcm 5 and the s 2 group
cooperatively improves decoding of both A- and G-ending codons.
36
Second position
U
C
Phe
U
Ser
(10)
(7) ncm 5Um
Leu
Pro
Leu
(4)
(3)a ncm 5U
Stop
(1)b
Stop
(2)a
Stop
(6)
Trp
Arg
His
(6)
(7)
(3) U
(10) ncm 5U
(9) mcm 5s2U
Gln
(1)b
(1)b
Ile
Thr
(13)
Ser
Asn
(11)
(4)
(10)
A
(2) Ψ
(4+5)c
Met
Val
(4) ncm 5U
(1)b
Ala
(14)
(7) mcm 5s2U Arg
Lys
(15)
G
(2)b
ncm 5U
(5) ncm 5U
(1)a
Gly
Asp
(11)
(11) mcm 5U
(14)
Glu
(2)a
(16)
(14) mcm 5s2U
(2)b
(3) mcm 5U
(2)a
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Third position
(1)a
C
G
Cys
(8)
(10)
Frist position
A
Tyr
(11)
Figure 8. Summary of the result presented in Paper II. Circles connected with a line
indicate one tRNA species. Transfer RNA species addressed in Paper II are labeled
with black or gray circles. Black circles connected with a line shows that a tRNA
species efficiently reads the indicated codons. Gray circles linked with a dashed line
indicate that a tRNA species reads the specified codons only when over-expressed.
The nucleoside at the wobble position is given for the U 34 containing tRNA species.
The number of genes coding for a tRNA species is indicated next to the circle for the
cognate codon, which also means that the primary anticodon sequence can be
deduced from the figure.
a
The gene(s) encoding the tRNA is nonessential.
gene(s) encoding the tRNA is essential.
37
b
The
Paper III: The Kluyveromyces lactis γ-toxin targets tRNA
anticodons.
To gain competitive growth advantage, various microorganisms produce and
secrete toxins. One example is the dairy yeast K. lactis, which secretes a
heterotrimeric toxin (zymocin), that causes growth arrest of sensitive yeast
cells (Gunge and Sakaguchi, 1981; For review, Schaffrath and Meinhardt,
2005; Sugisaki et al., 1983; White et al., 1989). Zymocin is composed of a α,
β, and γ subunit. The α and β subunits assist transfer of the cytotoxic
γ subunit into cells (Butler et al., 1991a; Gunge et al., 1981; For reviews,
Schaffrath and Meinhardt, 2005; Stark et al., 1990; Tokunaga et al., 1989).
Two classes of S. cerevisiae mutants resistant to zymocin have been
isolated (Butler et al., 1991a; Butler et al., 1994; For review, Schaffrath and
Meinhardt, 2005; Sugisaki et al., 1983; White et al., 1989). Class I mutants
are defective in binding and/or uptake of zymocin, as these mutants are
resistant to extra genius zymocin, but sensitive to intracellular γ-toxin. Class
II mutants are resistant to both extra genius zymocin and intracellular γ-toxin,
and therefore considered target mutants. Interestingly, strains deleted for any
of the ELP1-ELP6 or KTI11–KTI13 genes have been shown to be class II
mutants (Butler et al., 1994; Fichtner and Schaffrath, 2002; Frohloff et al.,
2001; Jablonowski et al., 2001b). As the Elongator complex was primarily
thought to function in elongation of Pol II transcription, it was assumed that
the γ-toxin was targeted to the Pol II transcription machinery in an
Elongator-dependent manner, and that the growth arrest was caused by
transcriptional inactivation (For review, Schaffrath and Meinhardt, 2005).
38
Previously, it was shown that elevated levels of the mcm 5 s 2 U 34 containing
tRNA Glu
suppressed the zymocin sensitivity of a wild-type S.
mcm 5s 2 UUC
cerevisiae strain (Butler et al., 1994). As all the class II mutants (elp1-elp6
and kti11-kti13) affect the synthesis of the mcm 5 group present in
tRNA Glu
, we hypothesized that the killer toxin-resistant phenotype
mcm 5s 2 UUC
induced by these mutations could be a consequence of the inability to modify
wobble uridine in tRNA Glu
.
mcm 5s 2 UUC
To test this hypothesis, we investigated the levels of tRNA Glu
in
mcm 5s 2 UUC
wild-type, elp3Δ, or trm9Δ cells after induction of intracellular γ-toxin. The
amount of tRNA Glu
was reduced with increased induction time in
mcm 5s 2 UUC
wild-type cells, whereas no reduction could be detected in elp3Δ or trm9Δ
cells. This supported the idea that the entire mcm 5 side-chain in
tRNA Glu
is required for the cytotoxicity of the γ-toxin. In addition to
mcm5s 2 UUC
ln
,
the tRNA Glu
, the mcm 5 side-chain is present in tRNA Gmcm
5s 2 UUG
mcm5s 2 UUC
Arg
, tRNA mcm
, and tRNA Gly
, but no reduction of these
tRNA Lys
5 UCU
mcm 5 s 2 UUU
mcm 5 UCC
tRNAs could be observed in wild-type cells after intracellular γ-toxin
expression, suggesting that these tRNAs are not substrates in vivo.
To investigate if γ-toxin acts directly on tRNA, we incubated total tRNA
isolated from wild-type, elp3, or trm9 cells with purified γ−toxin. Cleavage
Lys
ln
of tRNA Glu
, tRNA mcm
, and tRNA Gmcm
could be detected of
5 s 2 UUU
5s 2 UUG
mcm 5 s 2 UUC
tRNA
isolated
from
wild-type
cells
however,
tRNA Lys
mcm 5 s 2 UUU
and
ln
were cleaved with lower efficiency. The mcm 5 group is
tRNA Gmcm
5s 2 UUG
important for efficient cleavage in vitro, as reduced γ−toxin cleavage
efficiency was observed for tRNA isolated from elp3 or trm9 null mutants.
39
Analysis of the cleavage products revealed that the γ−toxin cleaves
Lys
ln
tRNA Glu
, tRNA mcm
, and tRNA Gmcm
5 s 2 UUU
5s 2 UUG
mcm5s 2 UUC
between position 34 and
35, generating a 2´, 3´ cyclic-phosphate and a 5´ hydroxyl-group. This
confirms that the γ-toxin acts directly on tRNA and suggests that
Lys
ln
, tRNA mcm
and tRNA Gmcm
might be γ-toxin targets
tRNA Glu
5 s 2 UUU
5s 2 UUG
mcm 5s 2 UUC
in vivo.
Consistent with the γ-toxin reactivity of the substrate tRNA in vivo and
in vitro, we showed that elevated levels of tRNA Glu
, but not
mcm5s 2 UUC
ln
or tRNA Gmcm
made a wild-type strain resistant to
tRNA Lys
5s 2 UUG ,
mcm 5 s 2 UUU
exogenous
zymocin.
However,
over-expression
of
tRNA Glu
mcm 5s 2 UUC
in
Lys
ln
combination with tRNA mcm
or tRNA Gmcm
or all three mcm 5 s 2 U 34
5 s 2 UUU
5s 2 UUG
containing tRNA species synergistically increased zymocin resistance. These
,
data suggests that all three mcm 5 s 2 U 34 containing tRNAs, tRNA Glu
mcm 5s 2 UUC
ln
, and tRNA Gmcm
, are γ-toxin substrates in vivo.
tRNA Lys
5s 2 UUG
mcm 5 s 2 UUU
Inhibition of protein synthesis arrests yeast cells in the G 1 phase of the
cell cycle (Hohmann and Thevelein, 1992; Johnston et al., 1977; Pyronnet
and Sonenberg, 2001; Unger and Hartwell, 1976; Wrobel et al., 1999).
Interestingly, zymocin also arrests sensitive yeast cells in the G 1 phase of the
cell cycle (Butler et al., 1991b). Therefore, we propose that the zymocin
induced G 1 arrest is most likely a consequence of a translational defect
caused by depletion or reduction of tRNA Glu
, tRNA Lys
, and
mcm5s 2 UUC
mcm 5 s 2 UUU
ln
tRNA Gmcm
.
5s 2 UUG
40
Summary:
In Paper III, we characterize the first eukaryotic toxin shown to target tRNAs.
ln
We show that all three mcm 5 s 2 U 34 containing tRNA species, tRNA Gmcm
,
5s 2 UUG
, and tRNA Glu
are cleaved by the γ-toxin between
tRNA Lys
mcm 5s 2 UUC
mcm 5 s 2 UUU
position 34 and 35. The γ-toxin resistance of elp1-elp6 and kti11-kti13
mutants can be explained by the fact that the these mutants are defective in
formation of the mcm 5 group present at position 34 which is required for
efficient cleavage (Figure 9).
mcm5s2 34
35 36
γ-toxin cleaves the three mcm 5 s 2 U containing
tRNA species between nucleoside 34 and 35
Glu
Lys
G ln
Figure 9. Model: K. lactis γ-toxin cleaves tRNA mcm
5 s 2UUC , tRNA mcm 5 s2UUU , and tRNA mcm 5 s 2UUG
between positions 34 and 35; the mcm 5 group at position 34 is important for efficient
cleavage.
41
3. Conclusions
Elevated levels of two tRNA species bypass the requirement for
the Elongator complex in transcription and exocytosis.
Lys
Increased expression of tRNA Gln
and tRNA mcm
5s 2 UUU
mcm5s 2 UUG
bypass the
requirement for the Elongator complex in transcription and exocytosis. This
observation suggests that the relevant function for the Elongator complex is
in tRNA modification (Paper I) (Figure 7).
Eukaryotic wobble uridine modifications promote a functionally
redundant decoding system.
The xm 5 side-chain present at the wobble uridine improves reading of
G-ending codons. The mcm 5 and s 2 groups cooperatively improve reading
of A- and G-ending codons (Paper II) (Figure 8).
The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
The γ-toxin cleaves the three mcm 5 s 2 U 34 containing tRNA species,
tRNA Gln
, tRNA Glu
, and tRNA Lys
between position 34 and
mcm5s 2 UUG
mcm5s 2 UUC
mcm5s 2 UUU
35. The γ-toxin resistance of mutants defective in formation the mcm 5 group
can be explained by the fact that this group is important for efficient cleavage
(Paper III) (Figure 9).
42
4. Acknowledgements
First, I would like to thank my supervisor Anders Byström for your passion in
research, and for your ability to create an educating environment. Special thanks for
all the wild ideas and discussions, and clearly showing me that the “crazy scientist”
still exists. Not to be forgotten, behind a wild scientist there is a hardworking and
helping wife, many thanks Mona for keeping him in line when needed and for the
many great dinners during the years. Thereafter I would like to acknowledge past
and present members of the AB group: Jian, Bo, Chen, Monica, and Marcus for
everything during these years. Moreover, I would like to thank Glenn, Mikael,
Tord, and their group members for the scientific discussions. I also would like to
thank P-A, Johnny, the dishwashing-, media-, and secretarial-personnel for
valuable services.
Special thanks to the “Innebandy people” for many excellent moments on and
off the court. It was great to know that Wednesdays was linked to entertainment,
laughter, and now and then some pain. Promise me to at least try to keep up the
good work even though, I know, it will be hard without me!
Outside the research field, I would like to acknowledge all my close friends:
Ante, Blomman, Mike, Nisse, Niklas, Erik and Carola, Tobbe, Maria, and Linea,
Magnus, Jessika and Petter, Per, Sussi, and Anna, Anna and Andreas, Niklas
and Soffan, Leif and Kristina, and many, many, more. A special thanks to “The
brothers” for not trying to understand my research. Nevertheless, all of you have
provided with everything close friends can and much more, thanks again.
Special thanks to my mother Birgitta, father Folke, brother Stefan, his sambo
Nina, and their lovely children Viktor and Emilia for being there and supporting
me when needed. I know, you know, how much you mean to me!
Finally, I would like to thank my life friend Maria for supporting me through
both the good and bad times. I hope that we have at least 70 more years to share and
enjoy together, love U.
Thank you all /Anders
43
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