Analysis of acceptor stem base pairing on tRNATrp aminoacylation and function in vivo

Marie Pak, Ian M. Willis, LaDonne H. Schulman

Research output: Contribution to journalArticle

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Abstract

The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNATrp was examined by complementation of an E. coli strain containing a temperature-sensitive tRNATrp gene (trpTts) and by monitoring aminoacylation levels in vivo. All derivatives of tRNATrp containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 °C). However, three acceptor stem derivatives (tRNATrp/ C1·G72, tRNATrp/C2·G71, and tRNATrp/A3·U70) required overexpression for growth at 42 °C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRN As appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNAUCATrp derivatives. These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNATrp. Substitution of the C1·A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNACCAfMet/G73) with A1·U72, the base pair found in tRNATrp, or G1·C72 resulted in the conversion of these tRN As into tryptophan-inserting elongator tRNAs in vivo. However, changes to U1·A72 or C1·G72 in tRNACCAfMet/G73 resulted in misaminoacylation and/or defects in translation. Our data indicate that the A1·U72 base pair is a context-dependent, negative identity element of tRNATrp.

Original languageEnglish (US)
Pages (from-to)2277-2282
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number3
StatePublished - Jan 21 1994

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Aminoacylation
Base Pairing
Derivatives
Transfer RNA
Tryptophan
Escherichia coli
Substitution reactions
RNA, Transfer, Met
Anticodon
Defects
Temperature
Discriminators
Electrophoresis
Transfer RNA Aminoacylation
Methionine
Urea
Genes
Gels
Acids
Monitoring

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of acceptor stem base pairing on tRNATrp aminoacylation and function in vivo. / Pak, Marie; Willis, Ian M.; Schulman, LaDonne H.

In: Journal of Biological Chemistry, Vol. 269, No. 3, 21.01.1994, p. 2277-2282.

Research output: Contribution to journalArticle

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