Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase.

M Weng,C A Makaroff,H Zalkin

doi:10.1016/s0021-9258(19)57252-0

Abstract

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.

Highlights

TheaminoacidsequenceofEscherichiacoli CTP esis to investigate the role of amino acid residues that are synthetase was derived from the nucleotide sequence important for glutamine amide transfer function [13,14,15]
The major 5’ end of pyrG CTP synthetase glutamine amide transfer domain contains mRNA was localized to a position approximately 48 three conserved regions similar to those in anthranilatesynbase pairs upstreaomf the translationinitiationcodon. thase, p-aminobenzoate synthase, GMP synthetase, and car
ThepyrG-eno transfer domain is located at the C-terminal end of the junction is characterized by multiple mRNA species molecule, encoded by DNA at the3’ end of the gene, consistwhich are ascribed to monocistronic pyrG and/or eno ent with a model [8]for evolution by gene fusion to augment mRNAs and a pyrG eno polycistronic mRNA

Summary

DISCUSSION

Sequences from NruI-BamHI and BumHI-PstI segments of the cloned DNA (Fig. 2C). The DNA sequence shown in Fig. 3 extendsfrom 11 bpupstream of theNruIsitetothe downstream PstI site at nucleotide 2442. The glutamine amide pyrG coding sequence, nuclease S1 mapping was repeated transfer domain is fused onto the C-terminalend of the using the 5’ end-labeled NruI-BumHI probe. Cise mapping was obtained by using a DNA sequencing ladder The conservation of amino acids in region 2 is sufficiently as a size standard (Fig. 5C) Theseresults confirm those high to predict that the conserved cysteine, residue 379 in obtained with restrictionfragment size standards, Corre- CTP synthetase, functions to form the covalent glutaminyl sponding sites for transcription initiation aroeverlined in Fig. intermediate as has been shown for Cys-84 in anthranilate synthase component I1 [14]. The same In a previous analysis of thepattern for fusion of the pattern of fragments was obtained when the nuclease S1 glutamine amide transfer domain to other protein chains, a concentration was increased 3-fold (data not shown). Theseresults indicate [8].Accordingto thismodel, after duplication, genes encoding

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I pMW I pMW5