A mutation in Escherichia coli tRNA nucleotidyltransferase that affects only AMP incorporation is in a sequence often associated with nucleotide-binding proteins.

L Q Zhu,H Cudny,M P Deutscher

doi:10.1016/s0021-9258(18)66797-3

L Q Zhu, H Cudny + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)66797-3

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Abstract

Escherichia coli strain 5C15 contains a mutation in the cca gene that decreases AMP incorporation by tRNA nucleotidyltransferase while leaving CMP incorporation unaffected. Earlier studies of the purified mutant enzyme suggested that the mutation was localized to the AMP-incorporating site. In order to analyze this mutation in more detail, the cca gene from strain 5C15 was cloned into plasmid pUC8. Analysis of tRNA nucleotidyltransferase activity in extracts of a strain transformed with this plasmid demonstrated an elevated level of CMP incorporation, but low AMP incorporation, as expected from the properties of the original mutant. Sequence analysis of the mutant cca gene revealed only a single G to A point mutation leading to a glycine to aspartic acid substitution at position 70 of the peptide chain. The amino acid change was localized to one of two Gly-X-Gly-X-X-Gly sequences present in the protein. This sequence has been identified previously near the nucleotide-binding domain of various proteins, but it has not been noted in enzymes that incorporate nucleotide residues. However, other sequences often associated with ATP-binding domains are not found in tRNA nucleotidyltransferase. The implications of these findings for our understanding of nucleotide-binding domains are discussed.

Highlights

Escherichia coli strain 5C15 contains a mutation in the cca gene that decreases AMP incorporation by tRNA nucleotidyltransferase while leaving CMP incorporation unaffected
Sequence present at the 3’ terminus of all tRNAs [1]. Accurate synthesis of this trinucleotide sequence proceeds despite the absence of any nucleotide material in the enzyme that might act as a template
We have previously described strains of Escherichia coli containing the isolation of mutant decreased levels of tRNA

Summary

Affects Only AMP Incorporation

Accurate synthesis of this trinucleotide sequence proceeds despite the absence of any nucleotide material in the enzyme that might act as a template [1] Previous studies from this laboratory led to the proposal that synthesis of the -C-C-A sequence derives from the specific arrangement of multiple donor and acceptor subsites within the active site of the enzyme including sites that recognize ATP, CTP, the 3’ terminal residues of tRNA, and the rest of the tRNA structure [2,3,4]. Our results indicate that the mutation in strain 5C15 is a single nucleotide change leading to a glycine + aspartic acid substitution at position 70 of the peptide chain This amino acid change is found in a peptide sequence, G-X-G-X-X-G, previously associated with nucleotide-binding domains of various enzymes (&lo), but this is the first example of its association with a nucleotide-incorporating site

PROCEDURES

RESULTS AND DISCUSSION

ATP crP