127
Endocytobiosis Cell Res. (2009) 19, 127-135
Analysis of the regulation of MatK gene expression
Reimo Zoschke1,
Linneweber1*
Oren
Ostersetzer2,
Thomas
Börner1,
Christian
Schmitz-
1
Institute of Biology, Humboldt University of Berlin, Chausseestr. 117, 10115 Berlin, Germany; 2Agricultural
Research Organization, Volcani Center, Bet Dagan 50250, Israel
*
Corresponding author: [email protected]
Abstract
Chloroplast genes of angiosperms contain
about 20 group II introns that require numerous nuclear-encoded factors for their removal
from precursor RNAs. Only one factor encoded by the chloroplast genome was suggested to be directly involved in splicing:
MatK. MatK shares sequence similarities with
maturases, bacterial splice factors encoded
inside their target introns. For one such maturase called LtrA a direct interaction with its
own mRNA resulting in translational repression has been demonstrated in a heterologous
expression system in E. coli. We used this
expression system to test the influence of the
expression of Zea mays MatK on the expression of a reporter gene under the control of
putative maize matK regulatory sequences.
We show that chloroplast-derived sequences
devoid of bacterial translational signals are
translated efficiently in E. coli. However, when
MatK is co-expressed with reporter gene constructs, no evidence for repression or activation of the reporter was detected, demonstrating that chloroplast MatK is not sufficient to
influence its own gene expression at least not
in this heterologous system.
Introduction
Chloroplasts are derived from cyanobacterial
endosymbionts. They have lost most of the
cyanobacterial genetic information with the
exception of a small set of genes coding
mostly for components of the photosynthetic
apparatus and for components of the chloroplast gene expression machinery. Among the
latter are genes for ribosomal RNAs and
tRNAs. Also, several dozen genes code for
protein subunits of the ribosome and three to
four genes code for subunits of an RNA polymerase. Thus, a substantial part of the
chloroplast transcriptional and translational
apparatus is encoded in its own genome
(SUGIURA et al. 1998). By contrast, factors
with a function in post-transcriptional processing of chloroplast RNAs like RNA splicing or
RNA editing are overwhelmingly encoded in
the nucleus and imported post-translationally
into the organelle (SCHMITZ-LINNEWEBER and
BARKAN 2007). In fact, more than a hundred
nuclear encoded RNA binding proteins have
been predicted to be localized in chloroplasts.
For some of them, roles for different chloroplast RNA processing steps have been determined (e.g. JENKINS and BARKAN 2001;
KOTERA et al. 2005; BEICK et al. 2008). Intriguingly, this wealth of nuclear encoded RNA
processing factors is countered only by a single putative chloroplast-encoded factor named
MatK potentially involved in chloroplast RNA
splicing.
MatK was originally identified based on
sequence homologies to mitochondrial maturases (NEUHAUS and LINK 1987), which are
involved in RNA splicing of precursor RNAs
containing group II introns. Group II introns
are highly structured ribozymes that have
been shown to self-splice in vitro (MICHEL and
FERAT 1995). In vivo, however, they require
proteinaceous auxiliary factors for splicing.
One class of such factors found for almost all
bacterial group II introns are RNA maturases.
128
Zoschke et al. – Analysis of the regulation of MatK gene expression
With few exceptions (WOLFE et al. 1992;
MOHR and LAMBOWITZ 2003; FUNK et al. 2007;
DUFFY et al. 2009; GAO et al. 2009), all known
maturase reading frames are located within
group II introns. In case of matK, the host intron is the trnK gene. The MatK sequence is
highly degenerated. Still, typical structural
features of maturase proteins have been identified based on secondary structure modeling
including the unique maturase RNA-binding
motif (domain-X) as well as a partial reversetranscriptase (RT) motif (BARTHET and HILU
2008). It has been shown that matK is expressed and that the encoded protein can be
detected in leaf tissue of various plant species
(BARTHET and HILU 2007). Indirect evidence
for a role of matK in splicing comes from plant
material with a compromised plastid translational apparatus. In these plants, a subset of
introns fails to be removed from precursor
RNAs (HESS et al. 1994; HÜBSCHMANN et al.
1996; VOGEL et al. 1997; VOGEL et al. 1999).
Possibly, the failure to express the MatK protein in this tissue is causing the splicing defect.
Direct functional evidence for a role of
MatK in splicing as well as studies on MatKRNA interactions are lacking. By contrast, it
has been demonstrated for the bacterial maturase LtrA that it binds with high affinity to domain IV of its host group II intron, a domain
that contains the maturase reading frame
(SINGH et al. 2002). The binding site has been
mapped to a sequence element containing the
ribosome binding site as well as the start
codon of the ltrA RNA. In the presence of
LtrA, this motif alone was sufficient to repress
translation of a reporter gene in vivo, probably
due to steric hindrance of ribosome entry
(SINGH et al. 2002). This suggests that bacterial maturases auto-regulate their expression
on a translational level. Contrasting this, we
show here by utilizing the same assay that
MatK can neither suppress the expression of
a reporter gene that was coupled to various
segments of the matK coding region nor to the
5’-UTR region. These data suggest that the
simple autoregulatory feedback loop of bacterial maturases has been lost in chloroplasts.
Results and Discussion
A conserved start codon in matK reading
frames from higher plants
The matK gene is transcribed in chloroplasts
and several RNA processing products have
been identified. It is however unclear, if all or
only a subset of RNA species produced from
the primary transcript are subject to translation. In particular, it is not known, whether
MatK is translated from unspliced trnK transcripts or whether translation occurs later,
after splicing. In order to narrow down the
regulatory element for matK translation, we
first tried to phylogenetically determine the
start codon of matK. We aligned matK sequences from a diverse set of angiosperms
(Figure 1). There are three alternative start
codons in the matK reading frame. The start
codon annotated in the original maize chloroplast sequence (gene bank acc. number
X86563), here called start 1, is likely wrong,
because our own sequencing efforts as well
as recent sequences from the database (gene
bank acc. number AY928077) report a sequence shorter by one base-pair: a Cnucleotide at position -37 to start 2 is missing
relative to the original Zea mays chloroplast
genome sequence submission. This deletion
occurs downstream of start 1 and thus would
obliterate the reading frame. Start codon 2 is
the one most commonly annotated in chloroplast matK sequences and is conserved in all
sequences from diverse plant species (Figure
1). Alternative start codon 3 is also found
throughout the alignment and is positioned 65
codons downstream of start 2. It seems however unlikely that the frame was conserved
from start 2 to start 3 across angiosperms if
start codon 2 was not a major translational
initiation site. It cannot be ruled out however
that start 3 is used as well.
Zoschke et al. – Analysis of the regulation of MatK gene expression
129
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
│-> pmZ3
CCCAAAGTCAAAAGAGCAATTGGCCTGCAAAAA------TAAAGGATTTGTT-CT-----CTTTTGTAATT-AGAACAAACATAAACAAATTGGATAG-TCCAAAGTCAAAAGAGCGATTGGCCTGCAAAAA------TAAAAAACTTGTT-CTGTTCTTTTTTGTAATTTAGAACAAACATAAACTAATTCGGTAG-CCCAAAGTCAAAAGAGCAATTGGCCTGCAAAAA------TAAAGGATTTGTT-CT-----CTTTTGTAATT-AGAACAAACATAAACAAATTGGATAG-TCCAAAGTCAAAAGAGCGATTGGCCTGCAAAAA------TAAAAGATTTGTT-CTTGTTTGTTTTGTAATT-AGAACAAACATAAACTAATTAGATAG-TCCAAAGTCAAAAGAGCGATTGGCCTGCAAAAA------TAAAAGATTTGTT-CTTGTTTGTTTTATAATT-AGAACAAACATAAACTAATTAGATAG-TCCAAAATCAAAAGAGCGATTGGGTTTAAAAAA------TAAAAGATTTCTAACCATCTTATTTTTTTATCTTATAAGATAAAAAACCAATTAGATGG--CCAAAATCAAAAGAGCGATTAGGGTGAAAAAA------TAAAGGATTTCTAAGTATTTTGTTATCCTATA---------CGAATATAAATTTTTCGTTT
TCCGAAGTCAAAAGAGCGATTAGGTTGAAAAAA------TAAAGGATTTCTAACCATCTTATTATCCTATAACACTATAACATAGACCAATTAAACGAAA
TCCAAAATCAAAAGAGCGATTGGGCTGAAAAAAAAAAAATAAAGGATTTCTAACTAGCTTGTT-----ATC---CTAGAA-------CATTTAGATGG-TCCAAAATCAAAAGAGCGATTAGGTTAAAAAAA------TAAAGGATTTCTAATAATCTTGTTTTGTTATT---CTATAATA-----TAACGAACAGA--
571
566
571
576
580
596
599
595
588
608
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
│-> pmZ2
start codon 1 ----------AAAA--GGAGCGGAAAAAGATCCGTGGATTAGACTCC-TTTTCTTCCCCAGGGGT--------TTGTATTAAAAA-------ATGCA------------AAAA--GGAGCGGAAAAAGATCCGTGGATTAGACTCC-CTTTCTTTCC--AGGGT--------TTGTATTAAAA--------ATGCA------------AAAA--GGAGCGGAAAAAGATCCGTGGATTAGACTCC-TTTTCTTCCCCAGGGGT--------TTGTATTAAAAA-------ATGCA------------AAAA--GGAGCGGAAAAAGTTCTATGGATTAGACTCC-CT-TTCTTTCC-TGGGT--------TTGTATT------------ATGCA------------AAAA--GGAGTGGAAAAAGTTCTATGGATTAGACTCC-CTCTTCTCTCC-TGGGT--------TTGTATT------------ATGCA------------AACA-----AAAATAGAGAGTCTGCTGATGAATTTACCCGTTATCCGAGGTATCTATTATTTCCT----------------AATAAAAT--AAATGG--AAAA--GAGAGGATAGAGGATCCGTTGATAAGTTTAC-CTGTATCCGAGGTATCTAT-TCGTTTA--------------TTACTAGAAT--CGAAAAAAAAAA--GAGATGATAGAGAATCCGTTGAGAAGTTTAC-CTGTATCCAAGGTATCTAT-TCTTACT-------------------AAAAT-----A----AAAA--GCGAGTTGAGAGAGTCCATTGATAGCTTTTATTTGTTTTCTGGGTATCTATATCATATTCGTTTTTTTTATTCGAATTCTAATTTA
--AA----CCTA--TTAAAGATAGAGAATCCGGTTAGCAGTCT-ACCTGTTTATGAGGTATCTATATC-TATAGCTTTCGT--------AGTCTAAT---
640
632
640
638
643
664
676
669
679
687
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
--------------ACACCCTGTTCTGACCATATTGTACTATGTATCACCATTTGATAAACCGAGAAATATTTC---TCTCTCTCTGATTCAAGTAGAAA
--------------ACACCCTGTTCTGACCATATTGCACTATGTATCATCATTCGATAAACCGAGAAATGCTTC---T-TCTCTCTGATTCAAGTAGAAA
--------------ACACCCTGTTCTGACCATATTGCACTATGTATCATCATTCGATAAACCGAGAAATGCTTC---TCTCTCTCTGATTCAAGTAGAAA
--------------ACACCTTGTTCTGACCATATTGCACTATGTATCATCATTTGATAAACCGAGAAATG--CT---TTTTTCTCTGATTCAAGTAGAAA
--------------ACACCTTGTTCTGACCATATTGCACTATGTATCATCATTTGATAAACCGAGAAATGGCTT---TTTTTCTCTATTTCAAGTAGAAA
----------------ACCCTGTTTTGACTGTATCGCACTATGTATCA---TTTGATAACCCAAGAAACCCTCTATTTTTACTTTTGTTTTCGGTCTAAA
----------------ACCTCGTTTTGACTATATCGCACTATGTTTCA---TTG-ATAACCCCCAAAATCCTC------TACCTTTAGTTCAACTAGAAT
----------------ACTTTGTTTTAACTGTATCGCACTATGTATCA---TTTGATAACCCTCAAAATCTTC------CGTCTTTGGTTCAAATCGAAT
TAATAGATAATAGAATATCCTGTTTTGACCATATCGCACTATGTATCA---TTTGATAATTCAACGAATCTCT-----GATCCTTTTGATCAAATAGAAT
----------------ACCTTATTTTGACTGTATCGCACTATGTGTCA---TTTCAGAACTCAAGAAAATAAAG-ACTTTACCTTCAGTTCAAATCGAAT
723
714
723
719
726
745
750
744
771
767
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
│-> pmZ1
--- start codon 2
TAC-----AAATGGAAAAATTCGAGGGTTATTCAGAAAAACTTAAATTTCCTAGACAATATTTCGTCT------ACCCACTACTCTTTCAGGAGTACATT
TAC-----AAATGGAAAAATTCGAAGGGTATTCAGAAAAACAGAAATCTCGTCAACAATACTTTGTCT------ACCCACTTCTCTTTCAGGAGTATATT
TAC-----AAATGGAAAAATTCGAAGGGTATTCAGAAAAACATAAATCTCGTCAACAATACTTCGTCT------ACCCACTTCTCTTTCAGGAATATATT
TAC-----AAATGCAAAAATTCGAAGGGTATTCAGAAAAACAGAAATCTCGTCAACACTACTTCGTCT------ACCCACTTCTCTTTCAGGAATATATT
TAC-----AAATGGAAAAATTCGAAGGGTATTCAGAAAAACAGAAATCTCGTCAACACTACTTCGTCT------ACCCACTTCTCTTTCAGGAATATATT
TTAAATTGAAATGGAAGAATTCCAAAGACATATAGAACTAGACAGGTCTTGGCAACATAATTTTTTAT------ATCCACTTATCTTTCAGGAATATATT
TTC-----AAATGGAGGAATTCCAAAGATATTTAAAGCTAAATAGATCTCAACAACACTACTTCTTAT------ATCCACTTATCTTTCAGGAGTATATT
TTC-----AAATGGAAGAAATCCAAAGATATTTACAGCCAGATAGATCGCAACAACACAACTTCCTAT------ATCCACTTATCTTTCAGGAGTATATT
TTCTA---AAATGGAGGAATATCAGTTATATTTGGAACTGGATAGATCTCGTCAACAGGACTTCCTAT------ACCCACTTGTTTTTCACGAATATATT
TTCAATCCAAATGGATAAATTTCAAGGATATTTAGAGTTCGATGGGGCTCGGCAACAGAGTTTTCTAT------ATCCACTTTTTTTTCGGGAGTATATT
812
803
812
808
815
839
839
833
862
861
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
TATGTGTTTGCCCATGATTATGGATTAAATGGTTCCG---------------AACTTGTCGAAATTATTGGTTCTAATAACAAGAAATTTAGTTCACTAC
TATGCATTTGCTCATGATTATGGATTAAACGGTTCTG---------------AACCTGTGGAAATAGTTAGTTGGAATAACAAGAAATTTAGTTCACTAC
TATGCATTTGCCCCTGATTATGGATTAAATGGTTCTG---------------AACCTGTGGAAATAGTTGGTTGTAATAACAAGAAATTTAGTTCACTAC
TATGCATTTGCTCATGATTATGGATTAAATGGTTCCG---------------AACCTGTGGAAATTTTTGGTTGTAATAACAAGAAATTTAGTTCAATAC
TATGCATTTGCTCATGATTATGGATTAAATGGTTCCG---------------AACCCGTGGAAATTTTTGGTTGTAATAACAAGAAATTTAGTTCACTAC
TATGCATTTGCATATGATCATGGTTTAAATAAATCGA---------------TTTTGTTGGAAAATTCAGGCA---AC---AGTAAATACAGTTTACTGC
TATGCACTTGCGCATGATCATGGTTTAAATAGAAATA---------GATCCATTTTTTTGGAAAATGTGGGTT---AT---AATAAATTCAGTTTACTAA
TATGCACTTGCTCATGATCATGGTTTAAATAGAAATA---------GGTCGATTTTGTTGGAAAATCCAGGTT---ATAACAATAAATTAAGTTTCCTAA
TACGGACTCGCTTATAGTCATGATTTAAATAGATCAA---------------TTTTTGTAGAAAATTTTGGTT---ATGATAATAAATATAGTTTACTAA
TATGTACTTGCTTATGATCATGGTTTAAATAGATTAAATAGAAATCGCTATATTTTCTTGGAAAATGCGGATT---ATGACAAAAAATATAGTTCACTAA
897
888
897
893
900
918
924
921
944
958
Os
Hv
Ta
Sof
Zm
Sol
Dc
Nt
Lj
At
--- start codon 3
TTGTGAAACGGTTAATGATTCGAATGTATCAG------CAGAATTTTTGGATTAATTTGGTTAATCATCCTAACCAAGATCGATTATTGGATTACAAC-TTGTGAAACGTTTAATTATTCGAATGTATCAG------CAGAATTTTTTGGATAACTCGGTTAATCATCCTAATCAAGATCGATTATTGGATTACAAA-TTGTGAAACGTTTAATTATTCGAATGTATCAG------CAGAATTTTTTGGATAACTCGGTTAATCATCCTAACCAAGATCGATTATTGGATTACAAA-TTGTGAAACGTTTAATTATTCGAATGTATCAG------CAGAATTTTTTGATTAATTCGGTTAATTATCCTAACCAAGATCGATTGTTTGATCACCGC-TTGTGAACCGTTTAATTATCCGAATGTATCAG------CAGAATTTTTTAATTAATTCGGTTAATTATCCTAACCAAGATCGATTGTTGGATCACCGT-TTGTAAAACGTTTAATTACTCGAATGTATCAA------CAGAATCATTTGATTCTTTCTGCTAATGATTCGAATCAAAATGTAATTTTGGGGCACAAGCA
TTGTGAAACGTGCAATTGAGCGAATGTATCAGTATGAGCAGAAGCATTTGATTCTTTTTGATAATGATTTTAACCAAAATAGATTTTTTGGACGCAAC-TTGTGAAACGTTTAATTACTCGAATGTATCAA------CAGAATCATTTTCTTATTTCTACTAATGATTCTAACAAAAATTCATTTTTGGGGTGCAAC-TTGTAAAACGTTTAATTACTCGAATGTATCAA------CAGAATCATTTGATTATTTCGGCTAATGATTCTAACAAAAATCGATTTTTGAGGTATAAT-TTACGAAACGCTTAATTTTGCGAATGTATGAA------CAGAATCGTTTGATTATTCCCACTAAGGATGTGAACCAAAATTCCTTTTTGGGGCATACC-insert A/B/C <-│
989
980
989
985
992
1012
1022
1013
1036
1050
Figure 1: Alignment of 5’-ends of matK genes from various angiosperms and position of construct inserts.
The 5’-region of matK DNA sequences from 10 different angiosperm species were aligned using Clustal X.
Conserved residues are shaded in black. Putative start codons are in red. The first and last nucleotides of
different inserts in constructs used in this study are indicated by vertical lines atop the alignment. The nucleotide missing in our own sequencing as well as in the latest Zea mays B73 chloroplast genome sequence
(AY928077) relative to the older Zea mays chloroplast genome sequence (NC_001666) is marked in bold
and crossed out (at -32 relative to start 2). Numbers are relative to the first nucleotide of the trnK-intron, in
which matK resides. Os = Oryza sativa; Hv = Hordeum vulgare; Ta = Triticum aestivum; Sof = Saccharum
officinalis; Zm = Zea mays; Sol = Spinacia oleracea; Dc = Daucus carota; Nt = Nicotiana tabacum; Lj = Lotus
japonicus; At = Arabidopsis thaliana
130
Zoschke et al. – Analysis of the regulation of MatK gene expression
Identification of MatK 5’UTR sequence
stretches capable of driving translation of
a reporter gene in E. coli
We next fused the sequence surrounding the
start codons 2 and 3, respectively, in frame to
a β-galactosidase (lacZ) reporter gene, deleting the endogenous lacZ start codon based on
a construct developed by SINGH et al. (Figure
2; SINGH et al. 2002). Transcription of the reporter gene constructs is driven by an arabinose-inducible promoter. We used three different constructs with varying 5’-UTR regions.
The matK insert in construct pmZ3 covers an
area from –730 to +259, where +1 is the first
nucleotide of matK start codon 2. Inserts in
construct pmZ1 and pmZ2 share the same 3’end with construct 3, but in construct 2 it extends 5’ only to position -132. Construct 1 carries the shortest insert that starts exactly at
start codon 2. The three 5’-matK-lacZ constructs pmZ1, pmZ2 and pmZ3 were transformed into recipient E. coli cells and plated
on arabinose- and X-Gal containing media, to
induce transcription and test the activity of
lacZ. None of the three inserts contains canonical shine-dalgarno sequences (ribosome
binding sites) in the vicinity of start codon 2 or
3. Nevertheless, all three inserts lead to blue
colonies, indicating that the respective matK
sequences are capable of driving lacZ translation (data not shown). E. coli harbors a versatile translation system that is capable of recognizing natural RNAs without any 5’-UTR
(VAN ETTEN and JANSSEN 1998) and tolerates
mutations in regulatory sequences like the
shine-dalgarno sequence (BARRICK et al.
1994). This could explain why sequence elements strongly deviating from standard bacterial 5’UTRs still work in vivo. The important
aspect for the analysis presented here is that
the heterologous sequences expressed from
our vectors are still a template for E. coli ribosomes.
Figure 2: Schematic map of reporter gene constructs used to test the interaction of MatK with ist own
mRNA. The lacZ start codon was replaced by three indepent inserts (pmZ1-3) of the matK 5‘-region comprising different start codons. M1-3 = three possible start codons. P = Arabinose-inducible promoter in front of
the lacZ gene
MatK expression does not inhibit translation of reporter gene constructs
With the proof of expression of our three 5’matK:lacZ constructs we next wanted to determine whether other factors can influence
translation of 5’-matK:lacZ fusions in trans.
We therefore generated an expression vector
for full-length Zea mays matK in order to test,
whether co-expression would lead to translational repression as seen for a bacterial matu-
Zoschke et al. – Analysis of the regulation of MatK gene expression
rase (SINGH et al. 2002). The matK reading
frame beginning with start codon 2 was fused
with the GST open reading frame under control of an IPTG-inducible promoter. This vector
called pmatM2pGEX was co-transformed with
our three 5’-matK:lacZ construct in E. coli
cells. The co-transformed cells were grown
and consecutively induced with first arabinose
and later with IPTG. As controls, the basic
RNA binding protein CYT-18 from Neurospora
crassa (KITTLE et al. 1991) and GST alone
were expressed in independent assays as
well. The activity of the lacZ gene was measured by standard spectrophotometric proce-
131
dures with and without induction of matK,
CYT18 or GST. Activities are expressed in
MILLER units (MILLER 1972) and are the mean
plus/minus standard deviation for 3 independent experiments (Figure 3). In contrast to previous observations made with the LtrA maturase from Lactococcus lactis (SINGH et al.
2002), we see no effect on the expression of
the reporter gene after the induction of
MatK:GST expression. Also, no effect relative
to the induction of GST alone was observed.
This holds true for all three constructs tested.
Steady-state levels of MatK:GST and GST
alone were tested by Western analysis using
Figure 3: Analysis of β-Galactosidase activity from 5’-matK/LacZ fusions in E. coli
Activity of β-Galactosidase LacZ reporter constructs pmZ1-3 containing different portions of the 5‘end of the
matK gene fused to the E. coli lacZ gene was assayed after co-transformation into E.coli with either
pmatM2pGEX, which expresses Zea mays MatK, with an empty expression vector (GST) or with the control
plasmid pEX560, which expresses the N. crassa CYT-18 protein. Compared are activities with induction
(+IPTG) or without induction (-IPTG). Activities are expressed in Miller units and are the mean ± the standard
deviation for three independent experiments.
a GST antiserum (Figure 4). Both proteins
were readily detectable after induction, but
MatK:GST accumulation was far lower than
that of GST alone. Also, small amounts of
both proteins were detected in uninduced
cells. MatK:GST and GST-Signals obtained
were similar for all three 5’-matK:lacZ reporter
constructs indicating a stable and reproducible
132
Zoschke et al. – Analysis of the regulation of MatK gene expression
protein expression after induction. Interestingly, while GST appears as a single signal in
immunoblot assays, MatK:GST is represented
as a ladder of bands ranging in size from
about 70 kD to bands of 25 kD, which is
equivalent to the mass of GST alone. The
calculated mass of full-length MatK:GST is 89
kD beginning at start codon 2 and 81 kD for
start codon 3. This exceeds the size of the
largest protein detected in Westerns. However, it is known that RNA binding proteins in
general and MatK in particular migrate faster
in SDS-PAGE gels than would be expected
from their size (LIERE and LINK 1995; VOGEL et
al. 1999; BARTHET and HILU 2007). It is therefore difficult to determine, which start codon
was used for matK translation. The various
bands found could indicate that different Ntermini are generated via alternative translation initiation. Alternatively, the complex protein pattern could be explained by proteases
attacking the MatK part of the MatK:GST fusion protein that is eventually degraded down
to the stable GST portion.
On the side, we see an unexpected, clear
positive effect on the expression of all 5’matK:lacZ fusions by CYT18 expression.
CYT18 is a splice factor for Neurospora group
I introns and accommodates both tRNAs as
well as introns via distinct binding surfaces. Its
versatility in RNA binding might lead to unspecific interactions with all 5’-matK:lacZ
RNAs. This could stimulate ribosome loading
by for instance dissolving secondary structure
elements.
The failure to see any alteration in βgalactosidase activity after expression of MatK
could mean that - in contrast to the Lactococcus maturase - MatK has lost the ability to
downregulate its own translation. There are
however a number of alternative explanations
for the failure to observe autoregulation of
MatK translation in our system that need to be
tested in the future. First, the fusion protein
might not be functional. No tertiary structures
are available for MatK, nor is homology to
better studied bacterial maturases high
enough to assess the influence of the GST tag
Figure 4: Immunological detection of GST:MatK and GST after co-expression with reporter gene constructs.
Cells co-transformed with reporter gene constructs and expression vectors pmatM2pGEX or pGEX-4T1 were
either induced with IPTG and arabinose (+) or not induced (-), grown for 18 hours and harvested. Aliquots of
cell suspensions were denatured and separated on a 12% polyacrylamid gel, blotted and probed with an
anti-GST antiserum. MlacZ indicates a control vector without any matK sequences, but with a full-length lacZ
reporter gene.
Zoschke et al. – Analysis of the regulation of MatK gene expression
on protein integrity. It is also unclear how
much protein is needed relative to the
5’matK:lacZ RNA in order to see an effect on
translation. For example, if we have a substantial excess of target RNA over MatK:GST
protein, we might not see any reduction in
translational efficiency due to limited RNAprotein repression complexes. Future quantifications of RNA and protein levels are required
to sort this out. Finally, MatK might only bind
to its own mRNA in the presence of a cofactor. Indeed, splicing in chloroplasts has been
demonstrated to rely on a multitude of different factors with MatK being only one of them
(SCHMITZ-LINNEWEBER and BARKAN 2007).
The identification of interacting proteins and
RNAs is therefore a pressing task for the further understanding of MatK. As a consequence, it will be important for the elucidation
of autoregulation to change from the bacterial
system with its limitations to an in planta system. For example, in situ overexpression of
partial matK sequences by stable chloroplast
transformation could help to identify an autoregulatory loop without altering the matK reading frame itself. Such an overexpression
would lead to the depletion of unbound MatK
and thus could free true mRNA for translation.
An increase in MatK protein amounts would
ensue. A working antibody to detect this is
available (BARTHET and HILU 2007).
Materials and Methods
Vectors
The reporter constructs pmZ1-3 are derived
from CapR pACYC184, which allows expression of lacZ fusions under the control of the
arabinose PBAD promoter (SINGH et al. 2002).
Each construct contains a different portion of
the matK 5’-region fused in frame to LacZ instead of the original Ll.LtrB DIVa sequence of
pACYC184 (see also Figure 1).
Maize MatK was expressed from pmatM2p
GEX. For cloning, the following oligos were
used: MatK_M2_F 5'- AAACCCGGTCGAC
133
AAATGGAAAAATTCGAAGGGTATMatK_R
5'- AAA AGC GGCCGCTCATTAATTAAGAGTAAGAGGATTCACCAGedF: 5' TGTGAAAG
AAATATTTTTCATTATTATAGTGGATCT
edR: 5' TTTTTCGAAGATCCACTATAATAAT
GAAAAATATTT MatK_M2_F5 and MatK_R
were used in a first PCR to amplify the entire
matK reading frame from maize DNA. A combination of primers edF and edR with
MatK_M2_F5 and MatK_R was used to
change a known maize editing site from C to
T. The final PCR-product was cloned into
pGEM-T by 'TA'-cloning and sequenced. To
release MatK, the vector was cleaved by SalI
and NotI and ligated into the same sites in
pGEX-4T1, leading to pmatM2pGEX.
LacZ reporter assays for translational
regulation by MatK
The reporter constructs were co-transformed
into E. coli BL21(DE3), which encodes an
IPTG-inducible T7 RNA polymerase, with the
AmpR pmatM2pGEX plasmid, which expresses the wild-type MatK protein from a T7
promoter or with the control plasmid pEX560,
which expresses the N. crassa CYT-18 protein (KITTLE et al. 1991) or with the pGEX-4T1
vector that expresses only GST. The transformants were grown on chloramphenicol (50
μg/ml) and ampicillin (100 μg/ml) on LB medium overnight and diluted 1:100 in fresh LB
containing the same antibiotics and incubated
at 20 C. At a culture density of A595=0.2, reporter gene expression was induced by 0.01%
(w/v) arabinose. Subsequently half of the culture was induced with 0.1 mM IPTG and, the
other half grew side-by side without induction.
After 18 hours, β-galactosidase activity was
assayed in triplicate, as described (Miller,
1972).
Immunoblot Analysis
Immunoblot analysis was carried out according to standard procedures (SAMBROOK et al.
1989) using a commercial anti-GST antibody
(Sigma).
134
Zoschke et al. – Analysis of the regulation of MatK gene expression
Acknowledgements
We thank Alan Lambowitz for vectors pACYC
184 and pEX560, Karsten Liere and Michi
Tillich for ciritical discussion of the data, and
Oliwia Makarewicz for help with establishing
the Miller assay. This work was supported by
an Emmy-Noether stipend to CSL.
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