Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS gene

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.     

DNA sequence of the Escherichia coli K-12 gamma-glutamyltranspeptidase gene, ggt.

The DNA sequence of ggt, the gene that codes for gamma-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12, has been determined. The sequence contains a single open reading frame encoding the signal peptide and large and small subunits, in that order. This result suggests that E. coli gamma-glutamyltranspeptidase is processed posttranslationally.     

DNA sequence of the gene coding for Escherichia coli ribonuclease H

The gene for Escherichia coli ribonuclease H has been studied by use of a plasmid which contains a segment of the E. coli chromosome. The genomic DNA was subcloned from pLC28-22 to pBR322 by use of various restriction enzymes. Such subcloning limited the RNase H gene to a piece of DNA no longer than 760 base pairs. Cells bearing plasmids containing the RNase H gene produce as much as 10-15 times the normal amount of RNase H without any drastic effect on maintenance of the plasmid or cell growth. DNA sequence analysis has permitted the prediction of a protein whose molecular weight is 17,559 (155 amino acid residues). The predicted sequence was confirmed by amino acid analysis, NH2-terminal amino acid sequence, and size determination of highly purified RNase H.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

DNA sequence of the araC regulatory gene from Escherichia coli B/r.

The DNA sequence of the araC regulatory gene from Escherichia coli B/r has been determined by the base-specific chemical cleavage reactions of Maxam and Gilbert. An open reading frame is found which codes for a protein of 292 amino acids. A nonsense mutation, araC5, is shown to result from a G to A transition at nucleotide 429 converting the tryptophan codon TGG to the amber codon TAG. A deletion which does not recombine with any known point mutation in araC, delta(araCO)719, removes all but the last 22 codons of the gene.     

Nucleotide sequence of the Escherichia coli mutH gene.

The complete nucleotide sequence of mutH gene from E. coli has been determined. Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein. Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene. Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced. One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).     

Mutational analysis of the Escherichia coli serB promoter region reveals transcriptional linkage to a downstream gene.

Genes encoding proteins with unrelated functions can be cotranscribed, and this may be used by cells to coordinate different metabolic pathways during growth. We describe a gene, designated sms, which is downstream from the serine biosynthetic gene serB in Escherichia coli but does not appear to be involved in amino acid (aa) biosynthesis. The sms gene is 1380 bp long. The Sms product migrates at 55 kDa on sodium dodecyl sulfate(SDS)-polyacrylamide gels and has a M(r) of 49472 (460 aa residues) calculated from the nucleotide sequence. The deduced Sms aa sequence shares regions of similarity with two ATP-dependent proteases, Lon and RecA, and contains two motifs: a C-x(2)-C-x(n)-C-x(2)-C motif, which is found in some nucleic acid binding proteins, and an ATP/GTP binding site motif. Insertional inactivation of sms led to increased sensitivity to the alkylating agent methylmethane sulfonate, but not to a requirement for serine or other metabolites. Several promoter mutations were isolated and characterized, which suggest that serB has a typical promoter recognized by sigma 70. After the serB coding sequence there is a 48-bp region with no obvious promoter sequence preceding the sms translation start codon. Analyses using sms'-lacZ fusions cloned downstream from wild-type and mutant serB promoters showed that sms is cotranscribed with serB.     

Nucleotide sequence of the Escherichia coli replication gene dnaZX.

The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization. The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III. The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger. The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins.     

Nucleotide sequence of the tag gene from Escherichia coli.

We have determined the complete nucleotide sequence of the tag gene, encoding 3-methyladenine DNA glycosylase I from Escherichia coli. From the nucleotide sequence it is deduced that the tag enzyme consists of 187 amino-acids and has a calculated molecular weight of 21.1 kdaltons. The tag enzyme is unusually rich in cysteine (8 residues) with a cluster of three consecutive cysteines near the C-terminal end. The tag coded DNA glycosylase does not show significant sequence homology to the alkA coded glycosylase in spite of that both of these enzymes catalyze the release of free 3-methyladenine from alkylated DNA.     

Identification and sequence of the drpA gene from Escherichia coli.

The drpA gene of Escherichia coli encodes a factor that is involved in global RNA synthesis. We establish that the drpA gene has been successfully cloned and describe the fine-structure map of three drpA-(Ts) mutations as well as the complete nucleotide sequence of the drpA gene. We identified a major sigma-70 promoter for the drpA gene on the bases of (i) its similarity to the consensus sequence and (ii) S1 protection and primer extension mapping data. In addition, the nucleotide sequence revealed a pair of dnaA boxes and a factor-independent terminator at the 5' end and 3' end of the gene, respectively. The deduced amino acid sequence of the DrpA protein showed a nucleotide-binding pocket found in some ATPases.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.