The asparaginyl‐tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316

Abstract

Escherichia coli strain N4316 is a mutant that exhibits temperature‐sensitive growth at 43°C and temperature‐sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called ‘rescue’, isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild‐type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature‐sensitive growth phenotype of the mutant at the non‐permissive temperatures. The cloned gene was sequenced, mapped to the 20.9‐min region of the E. coli chromosome and shown to code for a 466‐amino‐acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl‐tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read‐through of nonsense codons and protein copy number is discussed. Copyright © 1992, Wiley Blackwell. All rights reserved