JOURNAL OF BAcTERIOLOGY, Dec. 1992, p. 7819-7826 0021-9193/92/237819-08$02.00/0 Copyright X 1992, American Society for Microbiology
Vol. 174, No. 23
Role of Methionine and Formylation of Initiator tRNA in Initiation of Protein Synthesis in Eschenichia coli UMESH VARSHNEY ANnD UTTAM L. RAJBHANDARY* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 7 July 1992/Accepted 25 September 1992
We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU--CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vma,jKmaPP in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.
Initiation of protein synthesis in vivo always uses the amino acid methionine or its derivative formylmethionine (fMet) and a special tRNA, the initiator (14). In eubacteria, chloroplasts, and mitochondria, the initiator is used as fMet-tRNA. Following aminoacylation of the initiator tRNA (tRNAfMet), the methionyl-tRNA (Met-tRNAfmet) is formylated to fMet-tRNAf"et by Met-tRNA transformylase. The fMet-tRNA"et then binds to the P site on the ribosome in the presence of mRNA and initiation factors (8, 10). We recently developed an assay for studying the activities of mutant tRNAs in initiation in Escherichia coli (32). The assay measures the ability of a mutant initiator tRNA (T35A36) carrying an anticodon sequence change from CAU to CUA to initiate protein synthesis by using UAG as the initiator codon in a mutated chloramphenicol acetyltransferase (CAT) gene (CATaml.2.5). Mutant tRNA with this anticodon sequence change is now a substrate for E. coli glutaminyl-tRNA synthetase (GlnRS) (22, 24). Consequently, initiation from UAG occurs with fGln instead of fMet. Using this assay, we showed that mutations in the acceptor stem (Fig. 1) which affected kinetic parameters in formylation of the tRNAs in vitro also greatly reduced their activities in initiation in vivo (15, 31). These results indicated strongly that formylation of initiator tRNA is crucial for initiation, at least when the tRNA is aminoacylated with glutamine. In a continuation of these studies, we have asked whether overproduction of any E. coli protein can rescue the defect in initiation of the mutant tRNAs. We prepared a genomic library of E. coli DNA in a multicopy vector that carries one of the mutant tRNA genes (T35A36/G72) and the *
CATaml.2.5 gene and screened transformants which could grow on plates containing chloramphenicol. We have identified the gene which can rescue the activity of the mutant tRNA in initiation as that for E. coli methionyl-tRNA synthetase (MetRS) (1, 5). We have examined the effect of overproduction of MetRS on activities of other acceptor stem mutants which are defective in initiation and have found that some of the mutants can be rescued but others cannot. We have also studied the effect of overproduction of MetRS on levels of aminoacylation and formylation of the initiator tRNA mutants in vivo by using a gel electrophoresis method (11) which we described recently (30). These results show that an initiator tRNA mutant carrying fMet is a better initiator than one carrying fGln. In addition, the results provide a rationale for why overproduction of MetRS rescues the defect in initiation of some mutants but not of others. MATERIALS AND METHODS Plasmids and strains. Plasmid pRSV CATaml.2.5 trnfMT35A36/G72 (Ampr) was derived from pRSV CATaml.2.5 by inserting the T35A36/G72 mutant initiator tRNA gene between the PstI and BamHI sites (32). Similar plasmids carrying the other mutant tRNA genes have been described elsewhere (31). pBR322 plasmids carrying mutant tRNA genes were generated by subcloning a PstI-EcoRI fragment excised from M13mp8 (25) containing the tRNA genes into the PstI-EcoRI site of pBR322. Plasmids pACMS3 and pACMS5 were made by subcloning an -3.5kbp BssHII-PvuI fragment containing the E. coli MetRS gene into the EcoRI site of vector pAC1 (Kanr), where flanking ends of both the vector and the insert were converted into blunt ends by treatment with T4 DNA polymer-
Corresponding author. 7819
7820
VARSHNEY AND RAJBHANDARY AOH C C A - G73 A -G72 pTI - pC T2
Vol. 174, No. 23
Role of Methionine and Formylation of Initiator tRNA in Initiation of Protein Synthesis in Eschenichia coli UMESH VARSHNEY ANnD UTTAM L. RAJBHANDARY* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 7 July 1992/Accepted 25 September 1992
We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU--CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vma,jKmaPP in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.
Initiation of protein synthesis in vivo always uses the amino acid methionine or its derivative formylmethionine (fMet) and a special tRNA, the initiator (14). In eubacteria, chloroplasts, and mitochondria, the initiator is used as fMet-tRNA. Following aminoacylation of the initiator tRNA (tRNAfMet), the methionyl-tRNA (Met-tRNAfmet) is formylated to fMet-tRNAf"et by Met-tRNA transformylase. The fMet-tRNA"et then binds to the P site on the ribosome in the presence of mRNA and initiation factors (8, 10). We recently developed an assay for studying the activities of mutant tRNAs in initiation in Escherichia coli (32). The assay measures the ability of a mutant initiator tRNA (T35A36) carrying an anticodon sequence change from CAU to CUA to initiate protein synthesis by using UAG as the initiator codon in a mutated chloramphenicol acetyltransferase (CAT) gene (CATaml.2.5). Mutant tRNA with this anticodon sequence change is now a substrate for E. coli glutaminyl-tRNA synthetase (GlnRS) (22, 24). Consequently, initiation from UAG occurs with fGln instead of fMet. Using this assay, we showed that mutations in the acceptor stem (Fig. 1) which affected kinetic parameters in formylation of the tRNAs in vitro also greatly reduced their activities in initiation in vivo (15, 31). These results indicated strongly that formylation of initiator tRNA is crucial for initiation, at least when the tRNA is aminoacylated with glutamine. In a continuation of these studies, we have asked whether overproduction of any E. coli protein can rescue the defect in initiation of the mutant tRNAs. We prepared a genomic library of E. coli DNA in a multicopy vector that carries one of the mutant tRNA genes (T35A36/G72) and the *
CATaml.2.5 gene and screened transformants which could grow on plates containing chloramphenicol. We have identified the gene which can rescue the activity of the mutant tRNA in initiation as that for E. coli methionyl-tRNA synthetase (MetRS) (1, 5). We have examined the effect of overproduction of MetRS on activities of other acceptor stem mutants which are defective in initiation and have found that some of the mutants can be rescued but others cannot. We have also studied the effect of overproduction of MetRS on levels of aminoacylation and formylation of the initiator tRNA mutants in vivo by using a gel electrophoresis method (11) which we described recently (30). These results show that an initiator tRNA mutant carrying fMet is a better initiator than one carrying fGln. In addition, the results provide a rationale for why overproduction of MetRS rescues the defect in initiation of some mutants but not of others. MATERIALS AND METHODS Plasmids and strains. Plasmid pRSV CATaml.2.5 trnfMT35A36/G72 (Ampr) was derived from pRSV CATaml.2.5 by inserting the T35A36/G72 mutant initiator tRNA gene between the PstI and BamHI sites (32). Similar plasmids carrying the other mutant tRNA genes have been described elsewhere (31). pBR322 plasmids carrying mutant tRNA genes were generated by subcloning a PstI-EcoRI fragment excised from M13mp8 (25) containing the tRNA genes into the PstI-EcoRI site of pBR322. Plasmids pACMS3 and pACMS5 were made by subcloning an -3.5kbp BssHII-PvuI fragment containing the E. coli MetRS gene into the EcoRI site of vector pAC1 (Kanr), where flanking ends of both the vector and the insert were converted into blunt ends by treatment with T4 DNA polymer-
Corresponding author. 7819
7820
VARSHNEY AND RAJBHANDARY AOH C C A - G73 A -G72 pTI - pC T2
