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Molecular and Cellular Biology, September 1998, p. 5140-5147, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Harold J. Drabkin and Uttam L. RajBhandary*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 29 April 1998/Returned for modification 10 June 1998/Accepted 12 June 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein synthesis is initiated universally with the amino acid methionine (24, 43). Of the two species of methionine tRNAs found in all organisms, the initiator is used for initiation whereas the elongator is used to insert methionine internally. The codon used for initiation is almost always AUG, although in Escherichia coli and in other eubacteria, GUG and UUG are sometimes used. Using anticodon sequence mutants of E. coli initiator tRNA that are aminoacylated with amino acids other than methionine, it has been shown that while methionine is probably the best amino acid for initiation of protein synthesis in E. coli, glutamine, valine, phenylalanine, lysine, tryptophan and cysteine can also be used with various degrees of efficiency (6, 27, 36-38, 53, 56).

In eukaryotic systems, AUG is almost exclusively the codon used for initiation (25, 49). In mammalian cells, ACG and CUG have been found as initiator codons in a few mRNAs. A systematic analysis involving mutagenesis of the initiator AUG codon in the dihydrofolate reductase gene has shown that ACG, CUG, AUU, AUC, and AUA can all initiate protein synthesis to various degrees in vivo and in vitro (39). These mutant initiation codons differ from AUG by a single nucleotide. Because they use the wild-type initiator tRNA for initiation, protein synthesis in all of these cases is initiated with methionine. The pioneering work of Stewart et al. showed that in the yeast Saccharomyces cerevisiae also, AUG is almost exclusively the codon used for initiation (49). Cigan et al. have shown that AGG can be used as an initiation codon in yeast, if the mutant initiator tRNA with an anticodon sequence complementary to the AGG codon is also provided (7). They have further shown that UUG can be used to initiate protein synthesis, but only in yeast strains that carry mutations in either the alpha , beta , or gamma  subunit of the eukaryotic initiation factor eIF2 or the eukaryotic initiation factor eIF5 (9, 19). In both cases, protein synthesis is still initiated with methionine.

For functional studies of E. coli initiator tRNA in vivo, we previously described a strategy (42, 53) based on the use of mutant initiator tRNAs carrying an anticodon sequence change from CAU to CUA (designated the U35A36 mutant). The CUA anticodon sequence allows assessment of the initiator activity of the mutant tRNA in vivo by measuring the level of chloramphenicol acetyltransferase (CAT) expression from a reporter CAT gene which has UAG as the initiation codon (52, 53). Therefore, by coupling the U35A36 anticodon sequence mutation with mutations in the main body of the tRNA, the effect of the latter mutations on the overall activity of the mutant tRNA in initiation could be determined. Because the anticodon sequence of the E. coli initiator tRNA is a major determinant for aminoacylation with methionine, the U35A36 mutant initiator tRNA is now aminoacylated with glutamine (46, 47). Consequently, protein synthesis using these mutant tRNAs is initiated with formylglutamine.

Our objective is to develop an assay similar to this for studying the function of human initiator tRNA in mammalian cells. As in the case of the E. coli initiator tRNA, it is expected that most anticodon sequence mutants of mammalian initiator tRNA will be aminoacylated with an amino acid other than methionine. A previous study by Wagner et al. (55) had, however, shown that yeast initiator tRNA, aminoacylated with isoleucine in vitro with E. coli isoleucyl-tRNA synthetase, was inactive in initiation of protein synthesis because it bound extremely poorly to eIF2. This finding raised the possibility that protein synthesis in eukaryotic systems is initiated only with methionine.

This paper describes four different anticodon sequence mutants (CAUright-arrowCUA, CAUright-arrowCUG, CAUright-arrowCCU, and CAUright-arrowGAC, designated U35A36, U35G36, C35, and G34C36 mutants, respectively) of human initiator tRNA and studies on their activity in initiation in mammalian COS1 cells, using mutant CAT genes carrying UAG, CAG, AGG, and GUC as the initiation codons. The U35A36 and U35G36 mutant initiator tRNAs are not aminoacylated to any significant extent in COS1 cells by the endogenous aminoacyl-tRNA synthetases but can be aminoacylated with glutamine by coexpressing the E. coli glutaminyl-tRNA synthetase (GlnRS). The C35 mutant tRNA is aminoacylated in COS1 cells, at least partly with methionine, whereas the G34C36 mutant tRNA is aminoacylated with valine. We show that the U35G36 mutant initiator tRNA initiates protein synthesis with CAG as the initiator codon but extremely poorly. The U35A36 mutant tRNA does not initiate protein synthesis to any measurable extent. In contrast, the C35 and G34C36 mutant initiator tRNAs initiate protein synthesis quite well with AGG and GUC codons, respectively. These results suggest that glutamine is extremely inefficient in initiation in mammalian cells. In contrast, valine appears to function quite well as the initiating amino acid.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmids, tRNAs, and reporter genes. The mutant tRNAs described here are derived from the human initiator tRNA (15) and have mutations in the anticodon sequence. The C35 mutant human initiator tRNA gene with the CAUright-arrowCCU anticodon change has been described before (10). The U35A36 and the G34C36 mutant tRNA genes (anticodons CUA and GAC, respectively) were generated on M13mp7 by the phosphorothioate procedure (45). The mutant tRNA genes were cloned into the BglII site of the simian virus 40 (SV40) vector pSVBpUC (11). The U35G36 mutant tRNA gene (anticodon CUG) was generated on pSVBpUC by using the Stratagene Quickchange procedure and subsequently recloned into the BglII site of pSVBpUC.

The CAT reporter genes used contained either AUG, AGG, UAG, CAG, or GUC as the initiation codon. The CAT gene with the AGG1 mutation was constructed on an M13 vector carrying the wild-type Tn9 CAT gene, using the phosphorothioate procedure (45). In addition to the AUG-to-AGG initiation codon change, an upstream AGG codon was removed simultaneously during the mutagenesis. A HindIII/NcoI fragment of the CAT gene carrying the desired mutation was then excised and used to replace the corresponding fragment of the wild-type CAT gene in pRSVCAT. The CAT gene-containing plasmids pRSVCAT.2.5, am1.2.5 (53), and V1.2.5 (56) have been described previously and are referred to in this work as pRSVCAT AUG1, UAG1, and GUC1, respectively. The CAG1 mutation in the CAT gene was generated by the phosphorothioate method using an M13 vector and used to replace the wild-type CAT gene sequence in pRSVCAT to produce pRSVCAT CAG1.

For large-scale production of CAT proteins, the CAT AUG1, AGG1, and GUC1 genes were excised as HindIII/BamHI fragments and cloned into the pCDNA1 vector (Invitrogen).

The E. coli MetRS (54) and GlnRS (58) genes carried on pCDNA1 have been described before (10). The E. coli ValRS gene was cloned as an EcoRI fragment into the same site in pCDNA1 as the MetRS and GlnRS genes.

General procedures. Transfection of COS1 cells, preparation of extracts, and analysis of CAT activity were performed as reported earlier (10). Analysis of COS1 cell tRNA by acid-urea-polyacrylamide gel electrophoresis has also been described previously (10, 11). Oligonucleotides complementary to nucleotides comprising the anticodon stem and loop of the tRNA were used as hybridization probes.

Isolation of RNA from SV40-infected CV1 cells. SV40 virus stock carrying the G34C36 mutant human initiator tRNA was prepared from pSVBpUC as described before (10). Total tRNA was isolated from CV1 cells infected with SV40 carrying the G34C36 mutant initiator tRNA gene as described previously (10). Aminoacylation of tRNA with valine was performed using an E. coli S-100 extract enriched for ValRS as described by Wu and RajBhandary (57).

Measurement of rates of chemical deacylation of aminoacyl-tRNAs. The lability of the aminoacyl-tRNAs to base-catalyzed deacylation was monitored in two ways. [35S]Met-tRNA was prepared as described previously (12). A sample was adjusted to 0.2 M Tris-HCl (pH 9.6) and incubated at 37°C. At various times, an aliquot was withdrawn and adjusted to 5% trichloroacetic acid and chilled on ice. The trichloroacetic acid-precipitable counts remaining was determined by filtering on glass fiber filters followed by liquid scintillation counting of radioactivity on the filter.

Chemical deacylation rates of aminoacyl-tRNAs isolated from COS1 cells were measured by incubation of the tRNA with base followed by acid urea-gel electrophoresis to separate aminoacyl-tRNA from tRNA and Northern blot hybridization to detect the specific tRNA of interest (50). For the G34C36 mutant tRNA and the endogenous valine tRNA, total RNA from transfected COS1 cells was prepared as described previously (10). A 25-µl aliquot of the tRNA was adjusted to 0.2 M Tris-HCl (pH 9.6) and incubated at 37°C. At various times, a 5-µl aliquot was removed and mixed with an equal volume of 0.1 M sodium acetate (pH 4.5)-7 M urea-0.03% xylene cyanol-bromphenol blue, and the samples were frozen on dry ice. When all samples were ready, they were thawed and applied to 6.5% acid-urea gel (18, 50). After electrophoresis for approximately 20 h, the tRNA region was transferred to a Nytran Plus (Schleicher & Schuell) membrane. The tRNAs in the membrane were then hybridized to 5'-32P-labeled oligonucleotides complementary to either nucleotides 27 to 45 in the anticodon stem and loop of the G34C36 mutant initiator tRNA or nucleotides 13 to 33 in the D stem, loop, and anticodon stem of the endogenous valine tRNAs (as determined by use of the human valine tRNA sequences [2]). The amount of radioactivity in tRNA and aminoacyl-tRNAs was determined by quantitation with a PhosphorImager (Molecular Dynamics) and used to calculate the percentage of tRNA aminoacylated.

Isolation of CAT proteins for N-terminal sequence analysis. Each of 10 15-cm-diameter dishes of COS1 cells was transfected with 18 µg each of pCDNA carrying the CAT gene and pSVBpUC carrying the appropriate mutant initiator tRNA gene, using the DEAE-dextran/NuSerum method previously described (10, 23). Approximately 60 h posttransfection, the culture medium was removed and the cells were rinsed twice with phosphate-buffered saline. The cells were then lysed with 0.5% Nonidet P-40-50 mM KCl-Tris-HCl (pH 8) (1.8 ml/dish). After 5 min at room temperature, 1 ml of phosphate-buffered saline was added to each dish, the mixture was transferred to a centrifuge tube, and the samples were pelleted at 10,000 × g for 10 min. The supernatant was then mixed with 600 µl of chloramphenicol-caproate-Sepharose resin (Sigma) (59) equilibrated with 50 mM Tris-HCl (pH 7.8) and incubated at room temperature for 12 h with mixing. The material was then poured into a Pierce minicolumn, and the resin was washed with 50 mM Tris-HCl (pH 7.8) until the A280/ml of the effluent was less than 0.05. The column was subsequently washed with 50 mM Tris-HCl (pH 7.8) containing 0.3 M NaCl and then 1 M NaCl. The CAT protein was finally eluted with a solution containing 50 mM Tris-HCl (pH 7.8), 1 M NaCl, and 5 mM chloramphenicol. Elution was monitored by following CAT activity as measured spectrophotometrically (48). To 600 µl of 0.1 M Tris-HCl (pH 7.8), 22 µl of 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) was added along with 20 µl of 4 mM acetyl coenzyme A. An appropriate amount of enzyme dilution was added, and the mixture was used to blank a spectrophotometer at 412 nm. The reaction was initiated by addition of 10 µl of 10 mM chloramphenicol, and the A412 was measured at 3-min intervals. The fractions containing CAT activity were pooled, dialyzed against water, and concentrated by lyophilization. The sample was then applied directly to a sodium dodecyl sulfate-12% polyacrylamide gel. The proteins were then transferred from the gel to an Immobilon polyvinylidene difluoride membrane (Millipore), and the CAT protein was visualized by staining with Coomassie blue. The CAT protein band was then excised and used for N-terminal microsequencing.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The assessment of initiator tRNA function in mammalian cells is based on the ability of anticodon sequence mutants of initiator tRNA to initiate protein synthesis by utilizing mutant initiation codons in a reporter CAT gene (53). Figure 1 shows the four mutant anticodon-codon pairs of human initiator tRNA (15) and CAT reporter genes used in this study. The mutant initiator tRNAs with anticodon sequences CUA, CUG, GAC, and CCU are designated the U35A36, U35G36, G34C36, and C35 mutants, respectively. The corresponding mutant CAT genes are called CAT UAG1, CAG1, GUC1, and AGG1, respectively. Because protein synthesis in eukaryotes utilizes a scanning mechanism to locate the initiation codon (25), care was taken to ensure that there were no UAG, CAG, GUC, or AGG sequences upstream of the mutant initiation codon. In all cases, COS1 cells were cotransfected with three vector DNAs: pRSVCAT, pSVBpUC, and pCDNA1, carrying, respectively, the wild-type or mutant CAT reporter gene, the wild-type or mutant initiator tRNA gene, and the E. coli aminoacyl-tRNA synthetase gene (10, 11).


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  		FIG. 1.   Cloverleaf structure of vertebrate initiator tRNA. The anticodon sequence mutants and the mutant CAT reporter mRNAs designed to measure the activity of these tRNAs in initiation in vivo are shown.

Initiation of protein synthesis in mammalian cells with glutamine with UAG or CAG as the initiation codon. The U35A36 mutant E. coli initiator tRNA has been used extensively for studies on function of the initiator tRNA in E. coli (52, 53). This tRNA is aminoacylated with glutamine (46, 47) and initiates protein synthesis with UAG as the initiation codon (53). Therefore, as a first step to examine whether protein synthesis in mammalian cells could be initiated with glutamine, we generated an analogous mutant human initiator tRNA. It was expected that due to the anticodon mutation, the human initiator tRNA would no longer be a good substrate for MetRS but would become a substrate for the E. coli GlnRS. Figure 2 shows the results of assay for CAT activity in extracts of COS1 cells cotransfected with pRSVCAT UAG1, pSVB carrying the U35A36 mutant human initiator tRNA gene, and pCDNA carrying the E. coli GlnRS or MetRS gene (lanes 1 to 3). No CAT activity was detected in any of the extracts in assays using up to 60 µg of protein and 1 h of incubation. In contrast, extracts made from cells transfected with wild-type CAT gene contained substantial amounts of CAT activity (lane 7).


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  		FIG. 2.   Thin-layer chromatographic analysis of CAT activity in extracts of COS1 cells cotransfected with SV40-based plasmids carrying either the U35A36 (lanes 1 to 3) or U35G36 (lanes 4 to 6) mutant initiator tRNA gene, pRSVCAT UAG1 (lanes 1 to 3) or pRSVCAT CAG1 (lanes 4 to 6), and either pCDNA1 (lanes 1 and 4), pCDGlnRS (lanes 2 and 5), or pCDMetRS (lanes 3 and 6). Lane 7 is an extract from cells cotransfected with pRSVCAT AUG1, pSVBpUC, and pCDNA1. Lane 8 is an extract from a mock-transfected culture (no added DNA). The positions of mono- and diacetylchloramphenicols (Ac-CAM) and unreacted chloramphenicol (CAM) are indicated.

To assess the expression and aminoacylation levels of the U35A36 mutant tRNA, total tRNA was isolated from COS1 cells transfected with these plasmids, and the tRNA and aminoacyl-tRNA species were separated by electrophoresis on acid-urea-polyacrylamide gels (18, 50). The results of Northern blot analysis of such an experiment are shown in Fig. 3. The U35A36 mutant tRNA is expressed (lanes 4 to 6). As expected, it is not aminoacylated by the COS1 aminoacyl-tRNA synthetases (lane 4). The mutant tRNA is, however, aminoacylated in the presence of E. coli GlnRS but not in the presence of E. coli MetRS (compare lane 6 with lane 5), the extent of aminoacylation of the tRNA with glutamine being around 40%. These results show that the U35A36 mutant initiator tRNA is aminoacylated with glutamine in cells expressing the E. coli GlnRS but the aminoacyl-tRNA is not active in initiation of protein synthesis.


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  		FIG. 3.   RNA blot hybridization of tRNA isolated from COS1 cells cotransfected with SV40-based plasmids carrying the U35A36 (lanes 2 to 6) or U35G36 (lanes 7 to 11) mutant initiator tRNA gene and either pCDNA1 (lanes 4 and 9), pCDMetRS (lanes 5 and 10), or pCDGlnRS (lanes 6 and 11). A mixture of 5'-32P-labeled oligonucleotides complementary to nucleotides 27 to 45 of the mutant tRNAs were used as hybridization probes. Lanes 2, 3, 7, and 8 contain, respectively, the material in lanes 5, 6, 10, and 11 treated with 0.2 M Tris-HCl (pH 9.6) at 37°C for 20 min to deacylate the aminoacyl-tRNA. Lane 1 is a sample of RNA from mock-infected cells.

To examine whether the lack of activity of the CAT UAG1 reporter gene in initiation was because UAG, a stop codon, cannot be accommodated into the P site of a eukaryotic ribosome, the activity of the U35G36 mutant tRNA, also aminoacylated with glutamine, was tested by using the CAT CAG1 as the reporter gene. The CAT activity in extracts of COS1 cells cotransfected with these mutant genes is shown in Fig. 2. There is a weak but detectable activity in extracts from cells cotransfected with a vector carrying the E. coli GlnRS gene (lane 5) but not the MetRS gene (lane 6). The CAT activity seen in lane 5 of Fig. 2 corresponds to about 0.3% of that seen with the CAT AUG1 in lane 7. The poor activity of the U35G36 mutant initiator tRNA is not due to any problems in aminoacylation of the tRNA. Figure 3 shows that this mutant tRNA is aminoacylated to 60 to 70% in the presence of E. coli GlnRS (lane 11). There is also a small background of aminoacylation of this tRNA in the absence of any cotransfected E. coli aminoacyl-tRNA synthetase gene (lanes 9 and 10). This is probably due to aminoacylation of the U35G36 mutant initiator tRNA by the COS1 GlnRS.

Figure 3 also shows an interesting mobility difference between the U35A36 and the U35G36 mutant initiator tRNAs. This could be due to a difference in sequence or due to a difference in base modification in the anticodon loop of the mutant tRNAs (30), as a consequence of the presence of an A rather than a G at position 36. Modification of A37 in the anticodon loop to i6A is strongly dependent on the presence of A36 (4), which is present in the U35A36 but not in the U35G36 mutant tRNA.

Initiation of protein synthesis in mammalian cells with methionine, using AGG as the initiation codon. To rule out the possibility that non-AUG codons at the P site are intrinsically inefficient in mammalian cells, we constructed a C35 mutant human initiator tRNA corresponding to a mutant used in yeast by Cigan et al. (7). The corresponding yeast initiator tRNA mutant was shown to initiate protein synthesis at AGG codons by using methionine. The activity of the C35 mutant human initiator tRNA in initiation was measured by using CAT AGG1 as the reporter gene. The wild-type CAT sequence has upstream out-of-frame AGG codons, which were removed during the generation of the AGG1 mutation. Consequently, the CAT AGG1 reporter gene carries additional mutations upstream of the mutant initiation codon.

COS1 cells were transfected with the C35 mutant tRNA gene and pRSVCAT AGG1, in the presence of the pCD1 vector (lane 3) or of pCDMetRS (Fig. 4, lane 4). Extracts from cells transfected with the C35 mutant tRNA gene show significant CAT activity compared to cells in which the C35 mutant tRNA was absent (compare lanes 2 and 3). The CAT activity in cells expressing the C35 mutant tRNA and E. coli MetRS was even higher and close to the activity in cells expressing the wild-type CAT reporter gene (compare lanes 4 and 5). We have consistently observed an approximately twofold increase in CAT activity in extracts from cells which express E. coli MetRS compared to those which do not (compare lanes 3 and 4).


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  		FIG. 4.   Thin-layer chromatographic analysis of CAT activity in extracts of COS1 cells cotransfected with an SV40-based plasmid carrying either the wild-type (WT; lanes 2 and 5) or the C35 mutant (lanes 3 and 4) initiator tRNA gene, pRSVCAT AGG1 (lanes 2 to 4) or AUG1 (lane 5), and either pCDNA1 (lane 3) or pCDMetRS (lane 4). Lane 1 is an extract from mock-transfected cells. The positions of acetylchloramphenicol (Ac-CAM) and unreacted chloramphenicol (CAM) are indicated.

The aminoacylation levels of the C35 mutant initiator tRNA was studied by Northern blot analysis of the tRNAs separated on acid-urea-polyacrylamide gels (Fig. 5). Interestingly, PhosphorImager analysis of the tRNA expressed in these cells showed that the C35 mutant tRNA is about 60% acylated in the presence or absence of E. coli MetRS (Fig. 5, compare lanes 2 and 3). Thus, expression of E. coli MetRS increases the activity of the C35 mutant initiator tRNA in initiation (Fig. 4, lanes 3 and 4) without a corresponding increase in the extent of aminoacylation of the tRNA. This finding raises the possibility that in addition to methionine, the C35 mutant tRNA is aminoacylated with another amino acid, which functions poorly in initiation. Expression of E. coli MetRS leads to aminoacylation of more of the tRNA with methionine and an increase in initiation activity. A similar phenomenon in which overproduction of MetRS leads to aminoacylation of mutant tRNAs, ordinarily aminoacylated with glutamine or lysine, with methionine and thereby to increased activity in initiation has been noted before in E. coli (51, 54).


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  		FIG. 5.   RNA blot hybridization of tRNA isolated from COS1 cells cotransfected with an SV40-based plasmid carrying the C35 mutant initiator tRNA gene and either pCDNA1 (lane 2) or pCDMetRS (lane 3). The tRNA and aminoacyl-tRNA (aa-tRNA) species were separated on an acid-urea-polyacrylamide gel. A 5'-32P-labeled oligonucleotide complementary to nucleotides 27 to 45 of the C35 mutant tRNA was used as a hybridization probe. Lane 4, tRNA from mock-infected cells.

Initiation of protein synthesis in mammalian cells with valine, using GUC as the initiation codon. A mutant initiator tRNA with a valine anticodon (GAC) efficiently initiated protein synthesis from a GUC codon in E. coli (56). We therefore constructed the G34C36 mutant human initiator tRNA, which has a GAC anticodon. The data in Table 1 indicate that this mutant tRNA initiates protein synthesis from the GUC codon and produces 24 to 27% of the wild-type initiator tRNA CAT activity. The level of CAT activity does not change significantly in cells expressing E. coli MetRS or ValRS. There is a small but detectable activity with the CAT GUC1 in the absence of the G34C36 mutant initiator tRNA; however, CAT activity goes up by factors of 60 to 70 in the presence of the mutant tRNA. Analysis of the tRNA by acid-urea-gel electrophoresis followed by Northern blot analysis shows that the G34C36 mutant tRNA is essentially fully aminoacylated in vivo (>90% [data not shown]) by endogenous COS1 aminoacyl-tRNA synthetases.

                              
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  		TABLE 1.   Activity of the G34C36 mutant initiator tRNA in initiation in COS1 cells

Several lines of evidence suggest that the G34C36 mutant tRNA is aminoacylated in vivo with valine and that initiation with GUC utilizing this tRNA occurs with valine. First, total tRNA isolated from CV1 cells infected with SV40 carrying the G34C36 mutant initiator tRNA gene has an approximately fourfold increase in valine acceptance (Fig. 6B). Second, the rate of chemical deacylation of the G34C36 mutant aminoacyl-tRNA isolated from transfected cells is much lower than that of methionyl-tRNA and closely parallels that of valyl-tRNA (see Materials and Methods). Figure 6A shows a time course of deacylation of aminoacyl-tRNAs in 0.2 M Tris-HCl (pH 9.6) at 37°C. The half-life for deacylation of Met-tRNA is about 7 min, whereas the half-lives of the Val-tRNA and the aminoacyl-G34C36 mutant initiator tRNA are both about 53 min.


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  		FIG. 6.   (A) Rates of deacylation of aminoacyl-tRNAs in 0.2 M Tris-HCl (pH 9.6) at 37°C; (B) valine acceptor activity of tRNAs isolated from mock-infected CV1 cells and cells infected with SV40 carrying the G34C36 mutant initiator tRNA gene.

In attempts to obtain direct evidence that valine is used to initiate protein synthesis from the GUC codon, we isolated CAT protein expressed in COS1 cells transfected with the G34C36 mutant tRNA gene and the CAT GUC1 reporter gene carried on pCDNA1, using affinity chromatography on Sepharose columns containing covalently bound chloramphenicol (see Materials and Methods). As controls, we also isolated CAT protein expressed from pCDCAT AUG1 and pCDCAT AGG1. The pCD vectors were used for this work because these vectors replicate to very high copy numbers in the COS1 cells. The level of CAT proteins expressed in COS1 cells from pCDNA1 vector carrying the CAT AUG1 gene is approximately 10-fold the level expressed from pRSVCAT (data not shown). The expected sequences of the CAT proteins from the two reporters are shown in Fig. 7. The CAT protein isolated from cells carrying the wild-type CAT AUG1 gene appeared to be N-terminally blocked, presumably due to N-acetylation (Fig. 7A). When the CAT protein isolated from cells carrying the CAT AGG1 gene was analyzed in a similar manner, it was also found to be N-terminally blocked, consistent with the protein starting with methionine. In contrast, the CAT protein isolated from cells carrying the CAT GUC1 gene began with the second amino acid, aspartic acid (Fig. 7B). Thus, there must have been a different amino acid, most likely valine, preceding the aspartic acid residue. If it were methionine, it would also have been blocked at the N terminus.


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  		FIG. 7.   Predicted and determined N-terminal amino acid sequences of CAT reporter proteins isolated from transfected COS1 cells. Sequences of CAT AUG1 (A) and CAT GUC1 (B) are shown at the top.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This work provides the first example of initiation of protein synthesis in a eukaryotic cell with an amino acid other than methionine. We have shown that protein synthesis in mammalian cells can be initiated with codons other than AUG and with amino acids other than methionine. Codons such as AGG and GUC can be used to initiate protein synthesis quite well in the presence of the corresponding anticodon sequence mutants of the human initiator tRNA. While AGG initiates protein synthesis with methionine, GUC initiates most likely with valine. CAG can also be used to initiate protein synthesis with glutamine, although it is much poorer than AGG or GUC. Because initiation from CAG occurs only in cells expressing E. coli GlnRS (Fig. 2), initiation in this case must occur with glutamine.

While we have not proven that GUC initiates protein synthesis with valine, several lines of evidence suggest strongly that this is the case. First, overexpression of the G34C36 mutant initiator tRNA in CV1 cells infected with virus vector carrying the mutant tRNA gene leads to a fourfold increase in overall valine acceptance of total tRNA. Second, the rate of deacylation of the aminoacylated G34C36 mutant tRNA isolated from transfected COS1 cells closely parallels that of valyl-tRNA and is quite different from that of methionyl-tRNA. Valyl-tRNA and isoleucyl-tRNA have two of the most stable of the linkages between an amino acid and the tRNA (31). Third, N-terminal sequence analysis of the CAT protein produced by CAT GUC1 shows that it is not initiated with methionine. Given the fact that the G34C36 mutant tRNA is aminoacylated with valine and the synthesis of CAT is dependent on the presence of this tRNA, it is most likely that valine is used to initiate protein synthesis from the GUC codon. Therefore, in the discussion that follows, it is assumed that initiation from the GUC codon occurs with valine.

It is interesting that while the CAT protein initiated with methionine by using AUG and AGG as initiation codons is N-terminally blocked, presumably by an acetyl group (5, 35), valine is removed from the N terminus of the CAT protein initiated with GUC as the initiation codon. The finding of a N-terminal block in the CAT protein initiated with methionine is not surprising, since the expected N-terminal sequence is Met-Asp-Lys-, which is identical to that of the SV40 T antigen (44). The SV40 T antigen is known to be acetylated at the N-terminal methionine (34). It is not known at what stage valine is removed from the N terminus of the CAT protein initiated with a GUC codon. Normally, the enzyme methionine aminopeptidase (MAP) removes the initiating methionine from the growing polypeptide chain on the ribosome. Eukaryotic cells contain two methionine aminopeptidases (MAP1 and MAP2), and these enzymes are thought to be quite specific for methionine (1, 22). Also, MAP does not remove the N-terminal methionine when the penultimate amino acid is aspartic acid (35). Other nonspecific peptidases in mammalian cells that could remove valine from the CAT protein include leucine aminopeptidase (33). This enzyme is known to stop removing amino acids from the N terminus when the penultimate amino acid is basic, for example, lysine. Since the expected N-terminal sequence is Val-Asp-Lys-, this would explain why the CAT protein initiated with valine contains aspartic acid at the N terminus. It is not known whether valine is removed from the CAT protein in vivo or during the prolonged incubation of the extracts with the chloramphenicol affinity matrix during purification of the CAT protein. It should be noted that valine was missing from the N terminus of the CAT protein irrespective of whether the COS1 cell extracts were made in the presence or in the absence of a mixture of protease inhibitors.

While methionine and valine work quite well in initiation from non-AUG codons, initiation with glutamine from the CAG codon is extremely weak, and there was no initiation from an UAG codon. We cannot rule out the possibility that this is because the codons for glutamine start with a pyrimidine instead of a purine in AGG and GUC, although CAG (data not shown) and UAG work quite well in initiation in E. coli with the corresponding E. coli initiator tRNA mutants (53). Peabody has found that of the five non-AUG codons in the dihydrofolate reductase gene that initiate protein synthesis in CV1 cells, only one, CUG, starts with a pyrimidine (39). An alternate and, perhaps, more likely possibility is that the requirements for eIF2 binding to the aminoacylated initiator tRNA are fairly stringent and that glutamine with a polar side chain is not favored. This can be tested by overproducing the U35G36 mutant tRNA and studying its binding to eIF2 following its aminoacylation with glutamine.

In contrast to CAG, which is at least weakly active in initiation, there was no detectable activity with UAG. Both of the mutant tRNAs are aminoacylated with glutamine. The activities in vivo of an initiator tRNA mutant and of an mRNA containing a non-AUG codon depend on several factors: synthesis, stability and extent of aminoacylation of the tRNA, activity of the aminoacyl-tRNA in initiation and synthesis, and stability of the reporter mRNAs used. In yeast and in mammalian cells, a premature stop codon placed within an mRNA coding sequence can lead to decrease in mRNA stability and thereby to reduced mRNA levels (3, 41). Although UAG as the second or third codon in an mRNA can lead to reduced mRNA levels (60), it is unlikely that UAG as the initiation codon would have a similar effect, based on the mechanisms proposed.

Wagner et al. showed previously that the yeast initiator tRNA aminoacylated with isoleucine bound extremely poorly to the mammalian initiation factor eIF2 (55). This finding suggests that although isoleucine, like methionine, is hydrophobic, the branched alipathic side chain of isoleucine is detrimental to eIF2 binding. Alternatively, isoleucine affects the conformation at the 3' end of the initiator tRNA differently from methionine. It is therefore intriguing that valine is able to initiate protein synthesis. Since binding to eIF2 is a first step in the function of an initiator tRNA in initiation of protein synthesis (20), eIF2 must bind to the G34C36 mutant valyl-tRNA. It should be noted, however, that initiation with valine is not quite as good as initiation with methionine (Table 1), and it is possible that the binding affinity of eIF2 to the tRNA aminoacylated with valine is not as good as to the one aminoacylated with methionine. Measurements of binding affinity of the mutant initiator tRNAs to eIF2 are necessary.

Of the two non-AUG codons that we have shown to act as good initiation codons in COS1 cells, activity of the AGG codon is absolutely dependent on the presence of the corresponding mutant initiator tRNA. Activity of the GUC codon is also strongly dependent on the presence of corresponding mutant initiator tRNA (Table 1), although there is a low level of CAT activity in cells lacking the G34C36 mutant tRNA. The strict dependence on a mutant initiator tRNA for initiation from AGG suggests that the AGG codon cannot be translated by the wild-type initiator tRNA. This result is in agreement with the work of Cigan et al. (7) with S. cerevisiae and the work of Peabody (39) showing that of the many codons differing from AUG by a single nucleotide that were tested for the ability to function in initiation in CV1 cells, AGG did not initiate protein synthesis in vivo by using the wild-type initiator tRNA.

Finally, the strict requirement for a mutant initiator tRNA means that the CAT AGG1 gene and the C35 mutant initiator gene can, together, be used as an isolated pair to study the requirements for initiation in mammalian cells, without altering or affecting the activity of the endogenous initiator tRNA. This approach proved extremely useful for the study of structure-function relationships of E. coli initiator tRNA and the requirements for initiation in E. coli (42, 53). Thus, mutations at other sites in the initiator tRNA can be coupled to the C35 mutation to study the effects of these mutations on the overall function of the mutant tRNA in initiation, provided care is taken to assess the effects of the additional mutations on the steady-state levels of the tRNA and aminoacylation of the tRNA. The combination of the mutant CAT gene and the mutant initiator tRNA gene can also be used to study a variety of other interesting phenomena during initiation of protein synthesis (32). For example, Kozak (26) has shown that G at position +4 of a mRNA is important for efficiency of initiation in mammalian cells and for the formation of an initiation complex in vitro. Interestingly, unlike virtually every other tRNA, including the fungal initiator tRNAs, which have U33 preceding the anticodon sequence, vertebrate, plant, and insect initiator tRNAs have C33 (43). This finding raises the question of whether there is a fourth base pair between G at position +4 of the mRNA and nucleotide 33 of the initiator tRNA during initiation of protein synthesis in mammalian cells. This question can be addressed by mutation of the C33 in the C35 mutant initiator tRNA to U33 and mutation of the G at +4 on the CAT AGG1 gene to A and measuring CAT activity in extracts of cells transfected with different combinations of the mutant initiator tRNA and the mutant CAT genes. Other questions that may be addressed by using a reporter gene with AGG as the initiation codon instead of AUG, along with the C35 mutant initiator tRNA, include processes of leaky scanning (25), regulated use of alternate AUG codons for initiation (8, 14, 28), regulated use of alternate initiation codons such as CUG versus AUG as in the c-myc mRNA (16), use of internal ribosome entry sites (20, 21, 29, 40), ribosome shunting (13), and translational reinitiation (14, 17).

    ACKNOWLEDGMENTS

We thank M. Srinivasan for construction of the original CAT CAG1 reporter gene, X.-Q. Wu for preparation of an E. coli S-100 extract enriched for ValRS, Britt Persson for the suggestion of using chloramphenicol-caproate resin for purification of the CAT reporter protein expressed in COS1 cells, and William Shaw and Ralph Bradshaw for helpful discussions. We also thank Annmarie McInnis for her usual cheerfulness and care in preparation of the manuscript.

This work was supported by grants GM46942 and GM17151 from the National Institutes of Health.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, 68-671A, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-4702. Fax: (617) 252-1556. E-mail: bhandary{at}wccf.mit.edu.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Molecular and Cellular Biology, September 1998, p. 5140-5147, Vol. 18, No. 9
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