©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutants of Escherichia coli Initiator tRNA Defective in Initiation
EFFECTS OF OVERPRODUCTION OF METHIONYL-tRNA TRANSFORMYLASE AND THE INITIATION FACTORS IF2 AND IF3 (*)

Dev Mangroo (§) , Uttam L. RajBhandary (¶)

From the (1) Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 mRNA fMet-tRNA initiation complex in Escherichia coli. Overproduction of IF3 did not affect the initiator activity of any of the tRNA mutants studied.


INTRODUCTION

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)()(3) . The fMet-tRNA then binds specifically to the P site on the ribosome. This binding requires the initiation factors IF2 and IF3 (4) .

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.


MATERIALS AND METHODS

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.

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.

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.

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).

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.

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%.

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).

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 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).

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 ribosomemRNAfMet-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 30SmRNAfMet-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.

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.

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.

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) .

  
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



FOOTNOTES

*
This work was supported by Grant GM17151 from the National Institutes of Health (to U. L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

To whom correspondence should be addressed. Tel.: 617-253-4702; Fax: 617-252-1556.

The abbreviations used are: MTF, methionyl-tRNA transformylase; CAT, chloramphenicol acetyltransferase; GlnRS, glutaminyl-tRNA synthetase; MetRS, methionyl-tRNA synthetase; IPTG, isopropyl-1-thio--D-galactopyranoside; fGln, formylglutamine; PTH, peptidyl-tRNA hydrolase.

N. Mandal, D. Mangroo, and U. L. RajBhandary, unpublished results.

Mangroo, D., Limbach, J. A., McCloskey, J. A., and RajBhandary, U. L. (1995) J. Bacteriol., in press.

Drabkin and U. L. RajBhandary, unpublished data.


ACKNOWLEDGEMENTS

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.


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