Functional consequences of T-stem mutations in E. coli tRNA ThrUGU in vitro and in vivo

Margaret E. Saks, Lee E. Sanderson, Daniel S. Choi, Catherine M. Crosby, Olke C. Uhlenbeck

Research output: Contribution to journalArticle

Abstract

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNAThr UGU were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNAThr differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA ThrUGU T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNAThr can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.

Original languageEnglish (US)
Pages (from-to)1038-1047
Number of pages10
JournalRNA
Volume17
Issue number6
DOIs
StatePublished - Jun 1 2011

Keywords

  • Elongation factor Tu
  • tRNA gene knockout

ASJC Scopus subject areas

  • Molecular Biology

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    Saks, M. E., Sanderson, L. E., Choi, D. S., Crosby, C. M., & Uhlenbeck, O. C. (2011). Functional consequences of T-stem mutations in E. coli tRNA ThrUGU in vitro and in vivo. RNA, 17(6), 1038-1047. https://doi.org/10.1261/rna.2427311