Mutants of initiator tRNA that function both as initiators and elongators

U. Varshney, C. P. Lee, Baik Lin Seong, U. L. RajBhandary

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Abstract

We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU → CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C. P., Seong, B. L., and RajBhandary, U. L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli β-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such ''dual'' functions in tightly regulated expression of genes for proteins in E. coli.

Original languageEnglish
Pages (from-to)18018-18024
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number27
Publication statusPublished - 1991 Nov 8

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RNA, Transfer, Met
Transfer RNA
Escherichia coli
Mutation
Elongation
Galactosidases
Anticodon
Proteins
Terminator Codon
Glutamine
Kinetic parameters
Base Pairing
Nucleotides
Genes
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Varshney, U., Lee, C. P., Seong, B. L., & RajBhandary, U. L. (1991). Mutants of initiator tRNA that function both as initiators and elongators. Journal of Biological Chemistry, 266(27), 18018-18024.
Varshney, U. ; Lee, C. P. ; Seong, Baik Lin ; RajBhandary, U. L. / Mutants of initiator tRNA that function both as initiators and elongators. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 27. pp. 18018-18024.
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Varshney, U, Lee, CP, Seong, BL & RajBhandary, UL 1991, 'Mutants of initiator tRNA that function both as initiators and elongators', Journal of Biological Chemistry, vol. 266, no. 27, pp. 18018-18024.

Mutants of initiator tRNA that function both as initiators and elongators. / Varshney, U.; Lee, C. P.; Seong, Baik Lin; RajBhandary, U. L.

In: Journal of Biological Chemistry, Vol. 266, No. 27, 08.11.1991, p. 18018-18024.

Research output: Contribution to journalArticle

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N2 - We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU → CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C. P., Seong, B. L., and RajBhandary, U. L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli β-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such ''dual'' functions in tightly regulated expression of genes for proteins in E. coli.

AB - We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU → CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C. P., Seong, B. L., and RajBhandary, U. L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli β-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such ''dual'' functions in tightly regulated expression of genes for proteins in E. coli.

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