Evidence for misreading during tRNA-dependent protein synthesis in vitro

W. M. Holmes, G. W. Hatfield, Emanuel Goldman

Research output: Contribution to journalArticle

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Abstract

Leu-tRNA(Leu)-dependent protein synthesis has been examined in extracts of Escherichia coli cells which have a temperature-sensitive mutation in the leucyl-tRNA synthetase in order to assess the usefulness and fidelity of tRNA-dependent systems for studying codon-anticodon interaction. The results suggest that in MS2 RNA-directed reactions, there is a significant amount of misreading in the absence of added aminoacylated tRNA(Leu). By the incorporation of [35S]methionine or [14C]tyrosine, we estimate this level of misreading to be 15 to 30% of the amount of protein synthesized in samples reconstituted with several Leu-tRNA(Leu) isoaccepting species under these conditions. Examination of the synthesis of specific, known peptides spaced throughout the MS2 coat protein further suggests that in the absence of Leu-tRNA(Leu) the ribosomes manage to read through the CUC-encoded leucine at position 9 of the MS2 coat protein, and that in the presence of only Leu-tRNA3(Leu) or both (1 and 3)Leu-tRNA(Leu) (whose sequenced anticodons correspond to CUG and CUC codons, respectively), the ribosomes manage to read through the entire coat to the COOH terminus, including reading of the UUA-encoded leucine at position 86 of the MS2 coat. Beyond the endogenous misreading level, we have also examined the ability of purified Leu-tRNA(Leu) isoaccepting species to permit readthrough past the first leucine in an 'elongation' assay. These results argue that tRNA(4 and 5)(Leu) cannot read the CUC-encoded leucine at position 9, while tRNA(1 and 3)(Leu) can read this codon in vitro. Further, the unsequenced codon for the leucine residue at position 9 of the f2 coat protein would also seem to be of the CUX type. We conclude that tRNA-dependent systems should be regarded with caution for precise examination of codon-anticodon interaction, but such systems appear to be useful for certain kinds of experiments such as the elongation data reported here.

Original languageEnglish (US)
Pages (from-to)3482-3486
Number of pages5
JournalJournal of Biological Chemistry
Volume253
Issue number10
StatePublished - Dec 1 1978
Externally publishedYes

Fingerprint

RNA, Transfer, Leu
Transfer RNA
Codon
Leucine
Anticodon
Capsid Proteins
Proteins
Ribosomes
Elongation
Amino Acyl-tRNA Synthetases
Methionine
Escherichia coli
Tyrosine
In Vitro Techniques
Reading
Assays
RNA
Peptides
Mutation
Temperature

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Holmes, W. M. ; Hatfield, G. W. ; Goldman, Emanuel. / Evidence for misreading during tRNA-dependent protein synthesis in vitro. In: Journal of Biological Chemistry. 1978 ; Vol. 253, No. 10. pp. 3482-3486.
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title = "Evidence for misreading during tRNA-dependent protein synthesis in vitro",
abstract = "Leu-tRNA(Leu)-dependent protein synthesis has been examined in extracts of Escherichia coli cells which have a temperature-sensitive mutation in the leucyl-tRNA synthetase in order to assess the usefulness and fidelity of tRNA-dependent systems for studying codon-anticodon interaction. The results suggest that in MS2 RNA-directed reactions, there is a significant amount of misreading in the absence of added aminoacylated tRNA(Leu). By the incorporation of [35S]methionine or [14C]tyrosine, we estimate this level of misreading to be 15 to 30{\%} of the amount of protein synthesized in samples reconstituted with several Leu-tRNA(Leu) isoaccepting species under these conditions. Examination of the synthesis of specific, known peptides spaced throughout the MS2 coat protein further suggests that in the absence of Leu-tRNA(Leu) the ribosomes manage to read through the CUC-encoded leucine at position 9 of the MS2 coat protein, and that in the presence of only Leu-tRNA3(Leu) or both (1 and 3)Leu-tRNA(Leu) (whose sequenced anticodons correspond to CUG and CUC codons, respectively), the ribosomes manage to read through the entire coat to the COOH terminus, including reading of the UUA-encoded leucine at position 86 of the MS2 coat. Beyond the endogenous misreading level, we have also examined the ability of purified Leu-tRNA(Leu) isoaccepting species to permit readthrough past the first leucine in an 'elongation' assay. These results argue that tRNA(4 and 5)(Leu) cannot read the CUC-encoded leucine at position 9, while tRNA(1 and 3)(Leu) can read this codon in vitro. Further, the unsequenced codon for the leucine residue at position 9 of the f2 coat protein would also seem to be of the CUX type. We conclude that tRNA-dependent systems should be regarded with caution for precise examination of codon-anticodon interaction, but such systems appear to be useful for certain kinds of experiments such as the elongation data reported here.",
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Evidence for misreading during tRNA-dependent protein synthesis in vitro. / Holmes, W. M.; Hatfield, G. W.; Goldman, Emanuel.

In: Journal of Biological Chemistry, Vol. 253, No. 10, 01.12.1978, p. 3482-3486.

Research output: Contribution to journalArticle

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N2 - Leu-tRNA(Leu)-dependent protein synthesis has been examined in extracts of Escherichia coli cells which have a temperature-sensitive mutation in the leucyl-tRNA synthetase in order to assess the usefulness and fidelity of tRNA-dependent systems for studying codon-anticodon interaction. The results suggest that in MS2 RNA-directed reactions, there is a significant amount of misreading in the absence of added aminoacylated tRNA(Leu). By the incorporation of [35S]methionine or [14C]tyrosine, we estimate this level of misreading to be 15 to 30% of the amount of protein synthesized in samples reconstituted with several Leu-tRNA(Leu) isoaccepting species under these conditions. Examination of the synthesis of specific, known peptides spaced throughout the MS2 coat protein further suggests that in the absence of Leu-tRNA(Leu) the ribosomes manage to read through the CUC-encoded leucine at position 9 of the MS2 coat protein, and that in the presence of only Leu-tRNA3(Leu) or both (1 and 3)Leu-tRNA(Leu) (whose sequenced anticodons correspond to CUG and CUC codons, respectively), the ribosomes manage to read through the entire coat to the COOH terminus, including reading of the UUA-encoded leucine at position 86 of the MS2 coat. Beyond the endogenous misreading level, we have also examined the ability of purified Leu-tRNA(Leu) isoaccepting species to permit readthrough past the first leucine in an 'elongation' assay. These results argue that tRNA(4 and 5)(Leu) cannot read the CUC-encoded leucine at position 9, while tRNA(1 and 3)(Leu) can read this codon in vitro. Further, the unsequenced codon for the leucine residue at position 9 of the f2 coat protein would also seem to be of the CUX type. We conclude that tRNA-dependent systems should be regarded with caution for precise examination of codon-anticodon interaction, but such systems appear to be useful for certain kinds of experiments such as the elongation data reported here.

AB - Leu-tRNA(Leu)-dependent protein synthesis has been examined in extracts of Escherichia coli cells which have a temperature-sensitive mutation in the leucyl-tRNA synthetase in order to assess the usefulness and fidelity of tRNA-dependent systems for studying codon-anticodon interaction. The results suggest that in MS2 RNA-directed reactions, there is a significant amount of misreading in the absence of added aminoacylated tRNA(Leu). By the incorporation of [35S]methionine or [14C]tyrosine, we estimate this level of misreading to be 15 to 30% of the amount of protein synthesized in samples reconstituted with several Leu-tRNA(Leu) isoaccepting species under these conditions. Examination of the synthesis of specific, known peptides spaced throughout the MS2 coat protein further suggests that in the absence of Leu-tRNA(Leu) the ribosomes manage to read through the CUC-encoded leucine at position 9 of the MS2 coat protein, and that in the presence of only Leu-tRNA3(Leu) or both (1 and 3)Leu-tRNA(Leu) (whose sequenced anticodons correspond to CUG and CUC codons, respectively), the ribosomes manage to read through the entire coat to the COOH terminus, including reading of the UUA-encoded leucine at position 86 of the MS2 coat. Beyond the endogenous misreading level, we have also examined the ability of purified Leu-tRNA(Leu) isoaccepting species to permit readthrough past the first leucine in an 'elongation' assay. These results argue that tRNA(4 and 5)(Leu) cannot read the CUC-encoded leucine at position 9, while tRNA(1 and 3)(Leu) can read this codon in vitro. Further, the unsequenced codon for the leucine residue at position 9 of the f2 coat protein would also seem to be of the CUX type. We conclude that tRNA-dependent systems should be regarded with caution for precise examination of codon-anticodon interaction, but such systems appear to be useful for certain kinds of experiments such as the elongation data reported here.

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