The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.
Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees 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.
Aoki
H
H
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Yaworsky
P J
PJ
Patel
S D
SD
Margolin-Brzezinski
D
D
Park
K S
KS
Ganoza
M C
MC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism; D001226:Aspartate-tRNA Ligase; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017353:Gene Deletion; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature
D000596:Amino Acids; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D001216:Asparagine; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase