From the
We describe the effects of overproduction of methionyl-tRNA
transformylase and initiation factors IF2 and IF3 on the activity,
in vivo, of initiator tRNA mutants defective at specific steps
of the initiation process in protein synthesis. The activity of the
U35A36/G72 and U35A36/G72G73 mutants, which are defective in
formylation, was increased by overproduction of methionyl-tRNA
transformylase. In contrast, the activity of the C30:G40/U35A36 mutant,
which is formylated normally but is defective in binding to the
ribosomal P site, was not increased. Overproduction of IF2 had a strong
stimulatory effect on the activity of virtually all the mutants
carrying the U35A36 anticodon sequence change, including the U35A36,
U35A36/G72, U35A36/G72G73, and the C30:G40/U35A36 mutants. In cells
overproducing IF2, the amount of protein made by translation of a
mutant mRNA, which uses the U35A36 mutant initiator tRNA, is
severalfold higher than that made by translation of a wild type mRNA.
We discuss the possible implications of this result on overproduction
of proteins and on the order of assembly of the 30 S ribosome
Initiation of protein synthesis occurs universally with
methionine or its derivative formylmethionine. Of the two classes of
methionine tRNAs found in all organisms, the initiator is used
exclusively for the initiation of protein synthesis, whereas the
elongator is used for inserting methionine into internal peptidic
linkages
(1) . In eubacteria, mitochondria, and chloroplasts the
initiator tRNA is used as formylmethionyl-tRNA (fMet-tRNA)
(2) ,
whereas in the cytoplasm of eukaryotes, it is used as methionyl-tRNA
(Met-tRNA). Formylation of the initiator Met-tRNA is catalyzed by the
enzyme methionyl-tRNA transformylase (abbreviated here as
MTF)
The key features in the
Escherichia coli initiator tRNA important for formylation and
for targeting the fMet-tRNA to the P site on the ribosome have been
identified. The base-base mismatch at the end of the acceptor stem
unique to all eubacterial initiator tRNAs is important for formylation
of the tRNA and for preventing the tRNA from participating in
elongation and from being a substrate for peptidyl-tRNA
hydrolase
(5, 6, 7, 8, 9, 10, 11, 12, 13) .
The 3 consecutive G:C base pairs found in the anticodon stem of
virtually all initiator tRNAs are important for binding of the tRNA to
the P site on the ribosome
(14) .
Initiation factors are
thought to help ribosomes select initiator tRNA over other tRNAs at the
P site on the ribosome
(4) . However, the mechanism by which IF2
and IF3 facilitate this is not known. The presence of an N-blocked
amino acid such as formylmethionine, which is normally found only in
the initiator tRNA, is important for binding of the fMet-tRNA to
IF2
(15) . Whether other sequences in the initiator tRNA are also
important is not known. Protection experiments have shown that the
formylmethionine group, parts of the acceptor stem and the T stem, and
loop of the initiator tRNA are shielded by IF2
(16) . Similarly,
results of toeprint analyses indicate that parts of the anticodon stem
and loop sequence and/or structure are important for IF3 recognition of
the initiator tRNA
(17, 18) .
During work on
structure-function relationship of E. coli initiator tRNA, we
generated a number of tRNA mutants which are defective at specific
steps in the initiation pathway
(4) . This has allowed us to ask
whether the activity of the mutant tRNAs could be rescued by
overproduction of one or another component of the translational
machinery. For example, will overproduction of IF2 allow the
utilization of unformylated mutant initiator tRNA in initiation? Will
overproduction of IF3 increase activity of a tRNA defective at the step
involving binding to the ribosomal P site?
In this paper, we
describe the effect of overproduction of MTF and the initiation factors
IF2 and IF3 on activity of the various mutant initiator tRNAs. For
these studies, we use an in vivo assay for initiation
involving mutant tRNAs carrying an anticodon sequence change from CAU
to CUA and, as reporter, a mutant chloramphenicol acetyltransferase
(CAT) gene with UAG as the initiator codon instead of
AUG
(9, 19) . We show that overproduction of MTF rescues
specifically the activity of those mutants which are inactive in
initiation due to their being poor substrates for formylation but not
mutants which are defective at a later step in binding to the P site on
the ribosome. Overproduction of IF2 increases the level of CAT activity
in cells carrying all of the mutant tRNAs having the CUA anticodon.
Overproduction of IF3 had no effect on CAT synthesis with any of the
mutants tested.
Overproduction of MTF from
the dimeric construct (Fig. 1, right) was about
58-78-fold over normal levels, whereas that from the monomeric
construct was about 6-fold (data not shown). A similar observation was
made previously
(26) . The dimeric construct was used for most of
the experiments described here.
Overproduction of IF2 and IF3 was confirmed by immunoblot analysis
of cell extracts
(29, 30) and by Coomassie Blue staining
of protein gels. Both proteins are expressed to about 10-20% of
the total cell protein (data not shown).
Unlike
mutants carrying the U35A36 change, the wild type initiator tRNA is not
a substrate for GlnRS. Therefore, we also analyzed the effect of
overproduction of MetRS or MetRS and IF2 on CAT activity in cells
carrying the wild type tRNA or the U35A36 mutant. There was no
significant effect of overproduction of MetRS or MetRS and IF2 on
activity of the wild type tRNA. With the U35A36 mutant, there was an
approximately 3-fold increase in CAT activity in cells overproducing
MetRS alone. This is in agreement with previous results
(23) .
Overproduction of both MetRS and IF2 resulted in a further increase of
CAT activity in cells from 298 to 653%.
Overproduction of
IF2 results in a rather uniform increase in CAT activity of about
9-11-fold in cells carrying the various mutant initiator tRNAs
(). Further work is needed to understand this unexpectedly
``uniform'' increase in CAT activity. For the U35A36,
U35A36/G72, or the U35A36/G72G73 mutant tRNAs, the relative CAT
activities, respectively, of 905.2, 27.8, and 4.5% correlate well with
the activity of these tRNAs as substrates for Met-tRNA transformylase
(8, 11, 31). Therefore, the most likely explanation of the effect of
IF2 overproduction on CAT activities is increased utilization of the
formylated species of the mutant tRNAs in initiation rather than
utilization of the unformylated species of the tRNA.
A possible
explanation for the general stimulatory effect of overproduction of IF2
may lie in the nature of the amino acid attached to the mutant
initiator tRNAs. The mutant tRNAs with the U35A36 anticodon sequence
are normally aminoacylated with glutamine instead of methionine and,
therefore, carry fGln instead of fMet. Previous studies, based on
overproduction of MetRS in cells carrying the U35A36 mutant tRNA, have
shown that the U35A36 mutant tRNA carrying fMet is a better initiator
than the one carrying fGln
(23) . Thus, some component of the
translation machinery that acts on the initiator tRNA subsequent to its
formylation favors fMet over fGln. Since IF2 binds to the amino acid
acceptor end of the tRNA
(16) and requires an N-blocked amino
acid for binding
(15) , IF2 could have a lower affinity for tRNA
carrying fGln compared to tRNA carrying fMet. Therefore, the increased
activity of mutant initiator tRNAs, carrying the U35A36 change, in
cells overproducing GlnRS and IF2 could be due to increased utilization
of fGln-tRNAs in initiation. The finding that overproduction of IF2
also increases the activity of the Qi:2 tRNA is consistent with this
possibility. This tRNA, derived from E. coli glutamine tRNA,
is most likely aminoacylated with glutamine and is fully formylated
in vivo(10) .
It is not surprising that IF2 might
prefer fMet attached to the tRNA over fGln
(15) . This is the
case for most proteins which interact with aminoacyl-tRNAs. The E.
coli MTF prefers methionine over other amino acids, with a certain
order in its preference for the various amino acids Met > Gln >
Phe > Val, etc.
(8, 34) . The E. coli elongation factor EF-Tu prefers some amino acids over
others
(35, 36, 37) . The eukaryotic initiation
factor eIF-2 may have a very strong preference, if not an absolute
requirement, for methionine (38).
Another
possible explanation for the ``general'' enhancement in
activity of the mutant initiator tRNAs is an increase in levels of the
mutant tRNAs in cells overproducing IF2. An increase of
1.7-1.8-fold over levels of tyrosine tRNA, used as a control, was
observed for the U35A36 mutant (Fig. 6, compare lanes 2 with 3 and 4 with 5). However, this
increase in levels of mutant tRNAs can, at best, be only partly
responsible. First, it is not clear how the approximately 1.8-fold
increase in tRNA levels in cells overproducing GlnRS and IF2 can
account for the ``uniform'' 9-11-fold increase in CAT
activity. Second, most of the increase in the mutant initiator tRNA is
due to increase in a tRNA species (Fig. 6, band B`) that
is much less active in initiation.
With the U35A36
mutant tRNA, CAT activity in cells overproducing MetRS and IF2 is
somewhat lower than those overproducing GlnRS and IF2 (I).
This is in contrast to what is expected. Since fMet-tRNA is a better
initiator than fGln-tRNA
(23) , the activity of the U35A36 mutant
tRNA in cells overproducing MetRS and IF2 was expected to be higher or
at least the same as that in cells overproducing GlnRS and IF2. One
possible reason for this result is that the U35A36 mutant tRNA is an
extremely poor substrate for MetRS, V
A surprising result of this work is that CAT activity in
cells carrying the U35A36 mutant tRNA and overproducing MetRS, MetRS
and IF2, or GlnRS and IF2, is much higher than in cells carrying the
wild type tRNA and overproducing the same proteins (). A
3-fold increase in CAT activity was previously noted
(23) in
cells carrying the U35A36 mutant tRNA when MetRS was overproduced.
Current work confirms this and shows, additionally, that CAT activity
in cells carrying the U35A36 mutant tRNA and overproducing GlnRS and
IF2 can be as high as 9-fold over those carrying the wild type
initiator tRNA. The increased CAT activity is also reflected in the
amount of CAT protein made under these conditions (Fig. 5). The
mutant and wild type tRNA genes and the mutant and wild type CAT
reporter genes are expressed from the same plasmid and under similar
conditions. It is, therefore, unlikely that small differences in the
levels of expression of the tRNAs and/or CAT mRNAs account for the
large difference in CAT activity levels seen in cells carrying these
tRNAs.
The different CAT activities found in cells carrying the wild
type or the U35A36 mutant tRNAs could be an indication of the order of
assembly of the 30 S ribosome
Irrespective of the mechanism(s) involved, our
finding that the level of CAT expression from a UAG initiation codon is
much higher than that from the wild type AUG codon, especially when IF2
was overexpressed, opens up the possibility of overproducing proteins
initiated with glutamine and possibly other amino acids in E.
coli.
Finally, Gualerzi and co-workers
(41) showed
recently that excess IF3 repressed specifically the translation, in
E. coli cell-free extracts, of model mRNAs carrying AUU as the
initiation codon but not of mRNAs carrying AUG. Our finding that
overproduction of IF3 has no negative effect on translation of mRNAs
carrying UAG initiation codon in vivo (I) would
appear consistent with the notion that effect of IF3 is specific to
mRNAs with AUU as the initiation codon
(33) .
To
whom correspondence should be addressed. Tel.: 617-253-4702; Fax:
617-252-1556.
We thank Dr. John Hershey for the IF2 clone and the
anti-IF2 antibody, Dr. Cynthia Pon for the IF3 clone, Ira Schwartz for
the anti-IF3 antibody, Dr. Chan Ping Lee for synthesizing the
oligonucleotides, and Dr. Lisa Steiner for the use of Perkin-Elmer
thermoregulator. We also thank Dr. John Hershey, Dr. Paul Schimmel, Dr.
Al Dahlberg, Dr. Robert Zimmermann, and Dr. Bill McClain for comments
and suggestions on the manuscript and Annmarie McInnis for her
cheerfulness and care in the preparation of this manuscript.
mRNA
fMet-tRNA initiation complex in Escherichia coli.
Overproduction of IF3 did not affect the initiator activity of any of
the tRNA mutants studied.
(
)(3) . The fMet-tRNA then binds
specifically to the P site on the ribosome. This binding requires the
initiation factors IF2 and IF3
(4) .
Strains and Plasmids
The E. coli strains CA274 (HfrH lacZ125am trpEam)
(20) and
AA7852 (pth) (21) were described previously. Plasmid
pRSVCATam1.2.5 (Fig. 1, left) harboring the
mutant CAT gene and the various mutant tRNA genes
(19) ,
pACT7
(19) , pGFIBI
(22) , and pAC1 and its derivatives
containing the genes for GlnRS or MetRS
(19, 23) were
described before. Plasmid pAA2
(24) containing the IF2 gene and
pIM302
(25) harboring the IF3 gene were obtained from Drs. John
Hershey and Cynthia Pon, respectively. Plasmid pBluescript II SK+,
pET3d, and pACYC184 were obtained from Stratagene, Novagen, and New
England Biolabs, respectively.
Figure 1:
The
vectors used to co-express the CAT reporter gene and tRNA mutants
(left) and MetRS, GlnRS, MTF, or IF2 proteins in E. coli (right).
Cloning of the Gene for Methionyl-tRNA
Transformylase
The gene for MTF used in this study (designated
fmt) is on a DNA fragment which contains the gene for a
peptide deformylase (designated fms) upstream of the fmt gene
(26, 27) . The fms and fmt genes are part of an operon in E. coli and are
transcribed as a dicistronic mRNA
(28) . Two DNA fragments
containing the fmt gene were isolated by amplification of
E. coli chromosomal DNA using the polymerase chain reaction
method. The primers 5`-GATCGAATTCTCTAGAATAGAAGAAATAAT-3` and
5`-CTCTAAGCTTCATAAGTATAAAAACGCCCG-3` were used to isolate a
1.6-kilobase pair fragment containing the fms-fmt tandem
genes. Another primer, 5`-TACTGAATTCTAAGGATAAGAACTAACATG-3`, was used
to isolate a 1-kilobase pair fragment containing the fmt gene
alone. This primer also carried a mutation which changed the GUG
initiation codon in the fmt gene to AUG. Both fragments were
cloned into the EcoRI-HindIII site of the plasmid
pGFIBI. In these constructs, the fms-fmt and the fmt genes are under control of the E. coli lipoprotein
promoter.
Constructs for Overproduction of GlnRS, MetRS, MTF, IF2,
and IF3
The pACD vector used for overproduction of GlnRS, MetRS,
MTF, or IF2 (Fig. 1, right) was generated by replacing
the small AvaI-PvuII fragment of pACYC184 with an
820-base pair PvuII-SalI fragment from pBluescript II
SK+. Incompatible cohesive ends were filled in using the Klenow
fragment of DNA polymerase I in the presence of dNTPs. The 820-base
pair PvuII-SalI fragment contains a Lac promoter and
the Shine-Dalgarno and transcription termination sequences of gene
10 of bacteriophage T7. The transcription and translation signals
of gene 10 were excised from pET3d as an
XbaI-EcoRI fragment and cloned into the
XbaI-EcoRI sites in pBluescript II SK+.
GlnRS and MetRS
EcoRI fragments
containing the gene for GlnRS or MetRS were excised from pACQS or
pACMS
(23) , respectively, and cloned into the EcoRI
site of pACD. The resulting plasmids, pACDGlnRS and pACDMetRS, were
then used as vectors for the clon-ing of genes for MTF or IF2.
MTF
The fms-fmt gene was cloned into the
EspI site of pACD or pACDGlnRS. The vectors were digested with
CelII (an isoschizomer of EspI), the cohesive ends
were filled in and ligated to a PvuII fragment (see above)
from pGFIBI which contains the fms-fmt genes under the control
of the E. coli lipoprotein promoter. The monomeric fmt gene (see above) was excised from pGFIBI with AflIII and
PvuII and cloned into the NcoI (compatible with
AflIII) and CelII sites in the pACDGlnRS vector. The
CelII ends were filled in using the Klenow fragment of
DNA polymerase I. In this construct the fmt gene is under the
control of the lac promoter and the Shine-Dalgarno sequence
from bacteriophage T7 gene 10.
IF2
A 3.6-kilobase pair fragment containing the
gene for IF2 was excised from pAA2 with BamHI and
SalI and first cloned between similar sites in pBluescript.
The pBluescript-IF2 plasmid was digested with SphI and the
SphI ends were filled in using T4 DNA polymerase prior to
digestion with NotI. The large NotI-SphI
fragment containing the IF2 gene was then cloned between the
NotI and CelII sites of pACDGlnRS and pACDMetRS. The
CelII ends were filled in using the Klenow fragment of DNA
polymerase I. In both of these constructs, the IF2 gene is under the
control of the lac promoter.
IF3
The pACIF3 plasmid was constructed by excising
a 1-kilobase pair BamHI-EcoRI fragment containing the
IF3 gene from pIM302 and inserting it between the
EcoRI-SalI sites of pACT7. Incompatible ends, the
BamHI site in the fragment and the SalI site in the
vector, were filled in using the Klenow fragment of DNA polymerase I.
The IF3 gene is under the control of the lac promoter.
Growth of Cells
Transformants of E. coli CA274 and AA7852 (pth) strains were grown overnight
at 37 and 30 °C, respectively, in Luria-Bertani medium supplemented
with antibiotics as necessary. An aliquot of the overnight culture was
diluted 20-fold into 3 ml of fresh medium containing antibiotics as
required and IPTG, for full induction of IF2 and IF3 genes, and grown
for another 3-5 h (depending upon the strain, see legends to
figures) under shaking. For cells overproducing IF2, which grew
slightly slower, the overnight culture was diluted only 10-fold. No
significant difference was noted when a 20-fold dilution was used. When
required, ampicillin, kanamycin, tetracycline, and IPTG were added to
concentrations of 100 µg/ml, 20 µg/ml, 7.5 µg/ml, and 1
mM, respectively.
Preparation of Cell Extracts
Cells from 1.2 ml of
culture were pelleted by centrifugation for 10 min at 4 °C using a
Brinkman microcentrifuge and lysed as described
(9) . The cell
lysate was centrifuged for 10 min at 4 °C. An aliquot of the
supernatant was mixed with either 1 volume of CAT storage buffer (20
mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM
-mercaptoethanol, and 70% glycerol (w/v)) or 3 volumes of MTF
storage buffer (25.6 mM Tris-HCl, pH 8.0, 13.3 mM
-mercaptoethanol, 200 mM KCl, and 66.7% glycerol (w/v))
and stored at -20 °C.
Enzyme Assays
The activity of chloramphenicol
acetyltransferase (CAT), MTF, and -lactamase in cell extracts were
assayed as described
(9) . To minimize the effect of any changes
in pRSVCATam1.2.5 plasmid copy numbers, the specific
activities of chloramphenicol acetyltransferase are normalized to the
specific activities of
-lactamase in the same extract.
Analysis of the in Vivo Level of Formylation of
tRNA
Total tRNA was isolated under acidic conditions from E.
coli as described
(31) . The cells were resuspended in 300
mM sodium acetate, pH 4.5, 1 mM sodium EDTA, and
subjected to phenol extraction. Total RNA was precipitated with 2.5
volumes of cold ethanol. The precipitate was dissolved in 60 µl of
300 mM sodium acetate, pH 4.5, 1 mM sodium EDTA and
reprecipitated with 2.5 volumes of cold ethanol. The RNA was dissolved
in 20 µl of 10 mM sodium acetate, pH 4.5, 1 mM
sodium EDTA. An aliquot containing (0.0025-0.005 OD unit) was subjected to electrophoresis on a 6.5% urea
polyacrylamide gel at pH 5.0 and 4 °C. The tRNAs were transferred
to Nytran membrane
(31) , and the mutant initiator tRNAs were
detected using a probe complementary to nucleotides 29-47 or
40-56 of the U35A36 mutant tRNA. Uncharged tRNA markers were
prepared by incubating an aliquot of the isolated tRNA with an equal
volume of 100 mM Tris-HCl, pH 9.0, for 1 h at 37 °C.
General Methods
Protein concentration was
determined by the modified Lowry DC method as described by the supplier
(Bio-Rad) using IgG as the standard. Samples for SDS-polyacrylamide gel
electrophoresis were solubilized by boiling for 5 min in 62.5
mM Tris-HCl, pH 6.8, containing 4.0% SDS (w/v), 10% glycerol
(v/v), 10% -mercaptoethanol, and 0.02% bromphenol. Electrophoretic
transfer of proteins onto Immobilon membrane was performed for 2 h at
100 volts using a transfer solution composed of 25 mM Tris,
192 mM glycine, 20% methanol (v/v), and 0.01% SDS (w/v). The
pH of the solution was 8.3. For immunoblot analyses, bound antibodies
were detected using the ECL kit obtained from Amersham Corp. An Applied
Biosystems 380A DNA Synthesizer was used for the synthesis of
oligonucleotides. A Perkin-Elmer thermoregulator was used to carry out
the polymerase chain reaction.
RESULTS
Mutants of the E. coli Initiator tRNA
Fig. 2
(left) indicates the sites of mutations in the
initiator tRNA used in this study. The mutations in the main body of
the tRNA are coupled to those in the anticodon sequence from CAU to CUA
(U35A36). The CUA sequence allows the assessment of the initiator
activity of the mutant tRNAs in vivo by measuring the level of
CAT expression from a reporter CAT gene, CATam1.2.5, which has
UAG as the initiation codon
(9, 19) . Because of the
anticodon sequence change, the mutant tRNAs are aminoacylated with
glutamine. Therefore, protein synthesis from the CAT-am1.2.5 mRNA is initiated with formylglutamine (fGln). Based on amounts of
the CAT protein and CAT activity found in extracts, the U35A36
anticodon sequence mutant is almost as good an initiator as the wild
type tRNA
(9) . The G72 and G72G73 acceptor stem mutations make
the mutant tRNA extremely poor substrates for MTF in vitro and
in vivo(8, 9, 11, 31) .
Consequently, the U35A36/G72 and U35A36/G72G73 tRNAs are very poor
initiators
(9) . The C30:G40 and the U29C30A31:U39G40A41
anticodon stem mutants are good substrates for the formylating
enzyme
(8, 31) . However, the C30:G40/U35A36 and the
U29C30A31:U39G40A41/U35A36 mutants are poor initiators(
)
because of a defect in binding to the ribosomal P
site
(14) . The glutamine tRNA mutant, Qi:2, containing the
minimal features required for initiation such as formylation and P site
binding (Fig. 2, right) was shown previously to function
in initiation in vivo(10) , although not as well as the
wild type initiator tRNA.
Figure 2:
The nucleotide sequences of E. coli tRNA (left) and
tRNA
(right). The mutations studied
in tRNA
(left) are indicated by
arrows. The mutations in the acceptor and anticodon stems are
coupled to mutations in the anticodon sequence. The sites mutated in
tRNA
(right) to introduce the
minimal features required for initiation are indicated by
arrows.
The genes for the initiator tRNA mutants
U35A36, U35A36/G72, U35A36/G72G73, C30:G40/U35A36, and
U29C30A31:U39G40A41/U35A36, the glutamine tRNA mutant, Qi:2, and the
CATam1.2.5 reporter were carried on the pRSV vector
(Fig. 1, left).
Effect of Overproduction of MTF on the Activity of Mutant
tRNAs in Initiation
The activity of the U35A36 and
C30:G40/U35A36 mutant tRNAs in initiation was not significantly
affected by overproduction of GlnRS, MTF, or both GlnRS and MTF in
E. coli CA274 (). With the formylation defective
mutants (U35A36/G72 and U35A36/G72G73) also, overproduction of GlnRS
alone did not affect their activity in initiation. However,
overproduction of MTF led to increases in CAT activity in cells
carrying the U35A36/G72 and U35A36/G72G73 mutant tRNAs from 1.6 and
0.6% to 14.5 and 3.8%, respectively. When both GlnRS and MTF were
overproduced, there was a further increase in CAT activity to 66 and
8.0%, respectively. The increase in activity of these mutant tRNAs in
initiation depended upon the extent of overproduction of MTF. When the
extent of overproduction of MTF was reduced to 6-fold over normal
levels by using the pACD vector carrying the GlnRS gene and the DNA
fragment containing the fmt gene alone, CAT activity in cells
carrying the U35A36/G72 mutant increased only to about 10%, instead of
66% as found in cells overproducing MTF by about 60-fold (data not
shown).
Effect of Overproduction of MTF on Formylation of the
U35A36/G72 Mutant tRNA in Vivo
Total tRNA was isolated under
acidic conditions from E. coli CA274 transformants expressing
the U35A36 and U35A36/G72 initiator tRNA mutants and GlnRS or GlnRS and
MTF. The tRNAs were separated on a urea polyacrylamide gel and the
various forms of the initiator tRNA corresponding to the deacylated
tRNA (band A), formylaminoacyl-tRNA (band B), and aminoacyl-tRNA (band
C) were detected by Northern hybridization (Fig. 3). A probe for
the endogenous tyrosine tRNA (detected as bands D and E corresponding, respectively, to deacylated tRNA and
aminoacyl-tRNA) was used as an internal control to ensure that
approximately the same amount of total tRNA was analyzed. The U35A36
mutant tRNA was previously shown to be a good substrate for MTF. As
expected, this mutant tRNA is essentially quantitatively formylated,
irrespective of whether the cells are overproducing MTF or not (cf.Fig. 3
, lanes 2 and 3). In contrast, there
is no formylation of the U35A36/G72 mutant tRNA in cells overproducing
GlnRS alone (Fig. 3, lane 5). A very low level of
formylation of the U35A36/G72 mutant tRNA (band B) was
detected when GlnRS and MTF were co-expressed (Fig. 3, lane
6). This band is more discernible in films exposed for longer
periods (data not shown).
Figure 3:
Northern blot analysis of mutant initiator
tRNAs isolated from E. coli CA274 overproducing GlnRS or GlnRS
and MTF. The transformants were grown overnight at 37 °C in LB
containing 100 µg/ml ampicillin and 7.5 µg/ml tetracycline. An
aliquot (200 µl) was transferred to 4 ml of fresh medium containing
antibiotics and grown for 5 h. Total tRNA was isolated under acidic
conditions and an aliquot (0.005 OD) was subjected to
electrophoresis on a urea polyacrylamide gel at pH 5.0. The tRNA was
transferred to nytran membrane and the mutant initiator tRNAs were
detected with a
P-labeled oligonucleotide complementary to
nucleotides 29-47 of the U35A36 mutant initiator tRNA (Fig. 2).
A, B, and C indicate the location of the uncharged,
formylaminoacylated, and aminoacylated forms of the initiator tRNA
mutants, respectively; D and E indicate the location
of the uncharged and aminoacylated forms of tRNA
,
respectively.
Unlike the U35A36 mutant tRNA, the
U35A36/G72 mutant has a C1:G72 base pair. Introduction of a base pair
between nucleotides 1 and 72 in the initiator tRNA makes this tRNA a
good substrate for the PTH enzyme
(12, 32) . This could
account for the very low steady state level of formylation of the
U35A36/G72 mutant when both MTF and GlnRS were overproduced; that is,
the fGln-tRNA species produced was deacylated by the PTH enzyme to fGln
and tRNA. Analysis of the U35A36/G72 mutant tRNA isolated from an
E. coli strain AA7852 carrying a temperature-sensitive
mutation in the PTH enzyme confirms this possibility (Fig. 4). A
band corresponding to formylaminoacyl-tRNA (band B) was
observed when E. coli AA7852 expressing the U35A36/G72 mutant
tRNA, GlnRS, and MTF was grown at 30 °C (lane 3). This
band was not present when GlnRS alone was overproduced (lane
2). Inactivation of the PTH enzyme by further incubating the cells
at 37 °C resulted in most of the U35A36/G72 mutant tRNA being
formylated when GlnRS and MTF were co-expressed (lane 5),
whereas a very small amount of the tRNA was formylated when GlnRS alone
was overproduced (lane 4).
Figure 4:
Northern blot analysis of mutant initiator
tRNA isolated from E. coli AA7852 (pth) cells
overproducing GlnRS or GlnRS and MTF and grown at 30 or at 37 °C.
An aliquot (400 µl) of an overnight culture was transferred to 8 ml
of fresh medium containing antibiotics and grown for 3 h at 30 °C.
A portion of the culture (4 ml) was used to isolate total tRNA and
another portion was incubated at 37 °C for 3 more h prior to
isolation of total tRNA. An aliquot (0.01 OD
) of the tRNA
was subjected to electrophoresis on a urea polyacrylamide gel. The
mutant initiator tRNA was detected as described in the legend to Fig.
3. A, B, and C indicate the locations of the
uncharged, formylaminoacylated, and aminoacylated forms of the tRNA,
respectively.
Effect of Overproduction of IF2 on the Activity of Wild
Type and Mutant tRNAs in Initiation
As mentioned above,
overproduction of GlnRS alone had no significant effect on the activity
of any of the initiator tRNA mutants (). However,
co-expression of GlnRS and IF2 increased the activity of all of the
mutant tRNAs. Relative CAT activities in cells carrying the various
mutant tRNAs increased from 100 to 905.2% for the U35A36 mutant, from 3
to 27.8% for the U35A36/G72 mutant, from 0.4 to 4.5% for the
U35A36/G72G73 mutant, from 8.5 to 74% for the C30:G40/U35A36 mutant,
from 2.6 to 5.9% for the U29C30A31:U39G40A41/U35A36 mutant, and from
23.6 to 135.5% for the Qi:2 mutant. The activity of the initiator tRNA
with the wild type anticodon sequence CAU was not significantly
affected by expression of GlnRS alone or by co-expression of GlnRS and
IF2. Results of immunoblot analysis shown on Fig. 5confirm this
for the wild type and the U35A36 mutant tRNAs.
Figure 5:
Immunoblot blot analysis of CAT and
-lactamase levels in extracts from various E. coli CA274
transformants. Cell extracts were prepared from E. coli CA274
harboring the pRSV vector carrying the genes for either CAT2.5 and the wild type initiator tRNA or CATam1.2.5 and the
U35A36 mutant tRNA, and the pACD vector containing the genes for the
proteins indicated. An aliquot of the extract corresponding to 0.1 unit
of
-lactamase was subjected to electrophoresis on a 12%
polyacrylamide gel. The proteins were transferred to Immobilon membrane
and probed with anti-CAT (1:10,000) and anti-
-lactamase (1:20,000)
antibodies.
Overproduction of
GlnRS and IF2 has a much smaller effect on activity of the
U29C30A31:U39G40A41/U35A36 mutant tRNA. This is possibly because this
mutant, which lacks all three of the consecutive G:C base pairs, is
virtually inactive in binding to the ribosomal P site.
Effect of Overproduction of IF2 on the State of the
U35A36 Initiator tRNA Mutants in Vivo
In view of the dramatic
increase in CAT activity in cells carrying the U35A36 mutant and
overproducing IF2, we performed Northern blot analysis of the U35A36
mutant tRNA to determine whether overproduction of IF2 led to major
changes in the state of the tRNA (aminoacylation and/or formylation) or
in steady state levels of the tRNA. Some uncharged U35A36 mutant tRNA
was found in E. coli CA274 when MetRS was overexpressed
(Fig. 6, lane 2, band A), whereas very little uncharged
tRNA was found when GlnRS was overexpressed (lane 4). This is
as expected since the mutant tRNA is a better substrate for GlnRS than
it is for MetRS. In cells overexpressing MetRS and IF2 (lane
3), in addition to the uncharged tRNA (band A) and the
formylaminoacyl-tRNA (band B), a third species (band
B`) was observed. This species, also present in cells
overproducing both GlnRS and IF2 (lane 5), co-migrates with a
formylaminoacyl-tRNA species that lacks a base modification next to the
anticodon.(
)
Undermodification of the tRNAs could
be due to higher levels of expression of the U35A36 mutant tRNA in
cells overproducing IF2. Based on counts in bands hybridizing to the
mutant initiator tRNA (bands A, B`, and B) and to
tyrosine tRNA (band E), the amount of the mutant initiator
tRNA relative to the endogenous tyrosine tRNA went up on an average by
factors of 1.7-1.8 when IF2 was overexpressed (compare lanes
2 with 3 and 4 with 5). The
undermodified tRNA is less active in initiation of protein
synthesis.
Figure 6:
Northern blot analysis of mutant initiator
tRNA isolated from E. coli CA274 transformants overproducing
MetRS, GlnRS, MetRS, and IF2 or GlnRS and IF2. Transformants were grown
in medium containing 1 mM IPTG as described in the legend to
Fig. 3. An aliquot (0.0025 OD) of total tRNA was
subjected to electrophoresis on a urea polyacrylamide gel. The
initiator tRNA was detected with a
P-labeled
oligonucleotide complementary to nucleotides 40-56 (Fig. 2).
A and B indicate the location of the uncharged and
formylaminoacylated forms of the U35A36 mutant tRNA, respectively
whereas B` indicates the location of formylaminoacylated
U35A36 mutant tRNA lacking a base modification; D and E indicate the location of the uncharged and aminoacylated forms of
tRNA
, respectively.
Effect of Overproduction of IF3 on the Activity of
Wild Type and Mutant tRNAs in Initiation
I shows
the effect of overproduction of IF3 on CAT activity in cells carrying
the wild type or the mutant initiator tRNAs belonging to the various
classes. In view of indications that IF3 might recognize the three
consecutive G:C base pairs in the anticodon stem of initiator tRNA, it
was of interest to determine, in particular, the effect of
overproduction of IF3 on activity of the C30:G40/U35A36 mutant tRNA.
There was essentially no effect on the activity of the wild type or any
of the three mutants tested. Immunoblot analyses indicate that IF3 was
overproduced in these cells (data not shown). Coomassie Blue staining
of proteins fractionated on SDS gels indicate that IF3 was expressed to
about 10-20% of total cellular proteins in E. coli (data
not shown).
DISCUSSION
Effect of Overproduction of MTF
Formylation of
the E. coli initiator methionyl-tRNA is important for
initiation of protein synthesis
(5, 9) . In this work, we
have shown that the activity of tRNA mutants which are inactive in
initiation because of a defect in formylation can be partially rescued
by overproduction of MTF (). The effect of overproduction
of MTF is due to an increase in levels of formylation of the mutant
tRNAs (Fig. 4). Guillon et al. (11) also showed that the
initiation defect of mutant tRNAs could be partially rescued by
overproduction of MTF, although it was not established whether this was
due to increased formylation of the tRNA. The current work establishes
this for the U35A36/G72 mutant derived from the initiator tRNA and
further shows that overproduction of MTF rescues specifically the
activity of mutants which are defective in formylation.
Effect of Overproduction of IF2
In contrast to
results obtained with overproduction of MTF, overproduction of IF2 has
a more general effect on activity of virtually all of the mutant
initiator tRNAs carrying the U35A36 anticodon sequence change
(). The activity of the wild type initiator tRNA, however,
was not significantly affected by IF2 overproduction. The general
increase in activity of the mutant tRNAs carrying the U35A36 anticodon
sequence is not due to an increase in their formylation levels. 1)
There is no increase in the level of formylation of the U35A36/G72
mutant in E. coli AA7852 (pth) cells
overproducing IF2 (data not shown). 2) Overproduction of IF2 also leads
to a similar increase in activity of the U35A36 and the C30:G40/U35A36
mutant tRNAs. These tRNAs are fully formylated in vivo (Ref.
31; Fig. 6, lane 5).
(
)
for
aminoacylation down by a factor of more than 3,000
(39) .
Therefore, only a fraction of the tRNA is present as fMet-tRNA in cells
overproducing MetRS and IF2. Also, in contrast to cells overproducing
GlnRS and IF2, in cells overproducing MetRS and IF2, some of the U35A36
tRNA is uncharged (Fig. 6, lanes 2 and 3, band
A).
mRNA
fMet-tRNA pre-initiation
complex in vivo. Whether the 30 S ribosome, along with the
initiation factors, binds first to mRNA or to fMet-tRNA is not
established. It has been suggested that the 30 S ribosome binds to mRNA
or to fMet-tRNA in random order and in rapid equilibrium to form a
30S
mRNA
fMet-tRNA ternary complex
(4) . This is
thought to be followed by a rate-limiting ``rearrangement''
possibly involving the pairing of the initiation codon with the tRNA
anticodon. The increased activity in initiation of the U35A36 mutant
tRNA in cells overproducing MetRS MetRS, and IF2 or GlnRS and IF2 is
more easily understood if, under these conditions, translational
initiation of CAT mRNA proceeds mostly through the ordered binding of
the 30 S ribosomal subunit first to the initiator tRNA and then to the
mRNA. If the initiator tRNA binds first, ribosomes containing the
U35A36 mutant tRNA would only select mRNAs containing a UAG initiation
codon while those containing the wild type tRNA would select any of the
cellular mRNAs. Therefore, in cells overproducing the mutant initiator
tRNA, the mutant CAT mRNA containing the UAG initiation codon would be
translated preferentially. In contrast, in cells overproducing the wild
type initiator tRNA, the wild type CAT mRNA would have to compete with
other cellular mRNAs.
Effect of Overproduction of IF3
We showed
previously that the 3 consecutive G:C base pairs in the anticodon stem
of E. coli initiator tRNA were important for its binding to
the ribosomal P site
(14) . This binding is thought to be
facilitated in part by IF3
(17, 18) . Based on results of
toeprint analyses, Gold and co-workers
(17, 18) have
concluded that the 3 G:C base pairs, the sequence and/or the structure
of the anticodon loop, and the third nucleotide G of the initiation
codon are all important for IF3 selection of the initiator tRNA and the
initiation codon on the ribosome. Interestingly, overproduction of IF3
has no effect on the activity in initiation of the C30:G40/U35A36
mutant carrying a change in the 3 G:C base pairs or any of the other
initiator tRNAs tested (I). This result, which is
consistent with the conclusions of Gold and
co-workers
(17, 18) , suggests that the presence of 3
consecutive G:C base pairs in the anticodon stem of initiator tRNAs may
be a ``crucial'' requirement for its recognition by IF3.
Alternatively or additionally, 30 S ribosomes are already saturated
with IF3
(40) . Consequently, further increases in IF3 do not
have any effect.
Table:
Effect of overproduction of GlnRS, MTF, or GlnRS
and MTF on activity of mutant tRNAs in initiation
Table:
Effect of overproduction of GlnRS, MetRS,
GlnRS, and IF2 or MetRS and IF2 on activity of wild type and mutant
tRNAs in initiation
Table:
Effect of overproduction of IF3 on activity of
wild type and mutant initiator tRNAs in initiation
-D-galactopyranoside; fGln,
formylglutamine; PTH, peptidyl-tRNA hydrolase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.