Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs

Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs

Département de Biochimie, Université de Montréal, Montréal, PQ H3C 3J7, Canada

Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved December 4, 1997 (received for review September 23, 1997)

Abstract

The “cloverleaf” base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

Footnotes

↵ * V.B. and S.V.S. contributed equally to this work.
↵ † To whom correspondence should be addressed at: Université de Montréal, Département de biochimie, C.P. 6128, succursale Centre-Ville, Montréal, PQ H3C 3J7, Canada. e-mail: ceder{at}bch.umontreal.ca.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: mt, mitochondria; tRNA_su+ ^Ala, alanine suppressor transfer RNA; X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; DHFR, dihydrofolate reductase.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF003201–AF003233).