Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12.

(1) The nucleotide sequence of a 1991 bp segment of DNA that expresses the GMP reductase (guaC) gene of Escherichia coli K12 was determined. (2) This gene comprises 1038 bp, 346 codons (including the initiation codon but excluding the termination codon), and it encodes a polypeptide of Mr 37,437 which is in good agreement with previous maxicell studies. (3) The sequence contains a putative promoter 102 bp upstream of the translational start codon, and this is immediately followed by a (G + C)-rich discriminator sequence suggesting that guaC expression may be under stringent control (4) The GMP reductase exhibits a high degree of sequence identity (34%) with IMP dehydrogenase (the guaB gene product) indicative of a close evolutionary relationship between the salvage pathway and the biosynthetic enzymes, GMP reductase and IMP dehydrogenase, respectively. (5) A single conserved cysteine residue, possibly involved in IMP binding to IMP dehydrogenase, was located within a region that possesses some of the features of a nucleotide binding site. (6) The IMP dehydrogenase polypeptide contains an internal segment of 123 amino acid residues that has no counterpart in GMP reductase and may represent an independent folding domain flanked by (alanine + glycine)-rich interdomain linkers.     

Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12

Adenylosuccinate synthetase (EC 6.3.4.4), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis. The E. coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined. The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277. The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs. The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon. Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation. The purA control region did not resemble the control regions of the other known pur loci.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12

The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

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.     

Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12.

Porphobilinogen deaminase, the product of the hemC locus in Escherichia coli K12, catalyses the tetrapolymerisation of porphobilinogen (PBG) into the hydroxymethylbilane, preuroporphyrinogen. The hemC locus has been subcloned from the Clarke and Carbon plasmid pLC41-4. The sequence of the hemC structural gene and flanking DNA was determined by the dideoxy chain termination method of Sanger. The structural gene for hemC is located within a 942bp sequence encoding the monomeric PBG deaminase, molecular weight 33,857. The extent of the coding region was confirmed by sequencing the N-terminus of the purified enzyme and by determination of the molecular weight. The hemC locus is closely linked to the cyaA locus, the genes being transcribed in a divergent manner. Upstream of the hemC coding region, a possible promoter and three repeated GGATG sequences were identified. This is the first report of a complete DNA sequence for a structural gene specifying an enzyme of the heme biosynthetic pathway in prokaryotes.     

Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12

The nucleotide sequence of a 1.3-kb DNA fragment containing the entire pfkB gene which codes for Pfk-2 of Escherichia coli, a minor phosphofructokinase (Pfk) enzyme, is reported. The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an Mr of 33 000. Like other weakly expressed E. coli genes the codon usage in the pfkB gene is random; there is no strong bias for the usage of major tRNA isoaccepting species, and the codon preference rules of Grosjean and Fiers [Gene, 18 (1982) 199-209] are followed. This is the first report of the complete gene sequence of a phosphofructokinase.     

Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14-base-pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS-like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS-like sequence is homologous with other intercistronic sequences including that between the sdhB and sucA genes, the overall gene organisation being: sdhB-IS-sucAsucB-IS-. The patterns of codon usage indicate that sucB may be more strongly expressed than sucA, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex. The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceF gene product) indicates that each contains two analogous domains, an amino-terminal lipoyl domain linked to a carboxy-terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated approximately 100-residue lipoyl binding regions containing two short (approximately 19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one approximately 100-residue lipoyl binding region, with approximately 27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each approximately 300 residues, have an overall homology of 34% and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy-terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline-alanine segment is found approximately 50 residues into the subunit binding domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence of a regulatory region of the uidA gene in Escherichia coli K12

Summary Multiple regulatory events are involved in the expression of the uidA gene. A regulatory region of this gene has been located on a 460 base pair Sau3A-EcoRI fragment and its nucleotide sequence was determined by the dideoxy method using pEMBL plasmids. A preliminary analysis of this sequence revealed the presence of numerous palindromic structures with some overlaps.     

Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli.

We determined the DNA sequence of the hag gene of Escherichia coli K-12 and deduced the primary structure of the flagellin consisting of 497 amino acid residues. Comparison of the amino acid sequence with those of other bacterial flagellins revealed a high homology in the NH2- and COOH-terminal regions.