The nucleotide recognized by the Escherichia coli A restriction and modification enzyme

The nucleotide recognition sequence for the restriction-modification enzyme of Escherichia coli A (EcoA) has been determined to be GAG-7N-GTCA. This sequence is fairly similar, but distinctly different from the two other type I restriction enzyme recognition sites known for E. coli B and E. coli K12, respectively. N6-adenosine methylation has been observed at nucleotide positions 2 and 12 within that sequence after modification by EcoA. As a reference point for mapping the single EcoA site in lambda, the position of lambda point mutation Oam29 has been determined also.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

The DNA sequence on bacteriophage G4 recognized by the Escherichia coli B restriction enzyme.

Bacteriophage G4 possesses a single EcoB site located in the overlap between restriction fragments HinfI-12 and HaeIII-6. The sequence 5′-T-G-A … 8N … T-G-C-T occurs once in this segment and nowhere else in the DNA sequence of G4. Four independent G4 mutants that were not restricted by Escherichia coli B possessed the sequence 5′-T-G-A … 8N … T-G-C-C. The common sequence shared by the previously mapped EcoB sites on φXsB1, simian virus 40, f1, and fd DNAs is 5′-T-G-A … 8N … T-G-C-T … 9N … T. However, the sequence in the region of the G4 EcoB site contains an A instead of the final T conserved in these other examples. When the G4 EcoB site is aligned with the other EcoB sites, there are no conserved residues within 50 bases of the common sequence, 5′-T-G-A … 8N … T-G-C-T, except for those seven residues. The analysis of the EcoB site on G4 provides further evidence that only those seven bases are recognized by the E. coli B restriction enzyme.     

The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence

The EcoA restriction enzyme from Escherichia coli 15T- has been isolated. It proves to be an unusual enzyme, clearly related functionally to the classical type I restriction enzymes. The basic enzyme is a two subunit modification methylase. Another protein species can be purified which by itself has no enzymatic activities but which converts the modification methylase to an ATP and S-adenosylmethionine-dependent restriction endonuclease. The DNA recognition sequence of EcoA has an overall structure that is very similar to previously determined type I sequences. It is: 5'-GAGNNNNNNNGTCA-3' 3'-CTCNNNNNNNCAGT-5' where N can be any nucleotide. Modification methylates the adenosyl residue in the specific trinucleotide and the adenosyl residue in the lower strand of the specific tetranucleotide.     

The DNA sequence recognised by the HinfIII restriction endonuclease

HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:

$\text{5′-C-G-A-A-T-3′}\text{·····3′-G-C-T-T-A-5′}$

In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.     

The nucleotide sequence and restriction enzyme sites of the polyoma genome.

We present the 5295 nucleotide-long sequence of the polyoma genome and the restriction enzyme digestion sites predicted from this sequence.     

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.     

The distribution of restriction enzyme sites in Escherichia coli.

A statistical analysis of physical map data for eight restriction enzymes covering nearly the entire genome of E. coli is presented. The methods of analysis are based on a top-down modeling approach which requires no knowledge of the statistical properties of the base sequence. For most enzymes, the distribution of mapped sites is found to be fairly homogeneous. Some heterogeneity in the distribution of sites is observed for the enzymes Pstl and HindIII. In addition, BamHI sites are found to be more evenly dispersed than we would expect for random placement and we speculate on a possible mechanism. A consistent departure from a uniform distribution, observed for each of the eight enzymes, is found to be due to a lack of closely spaced sites. We conclude from our analysis that this departure can be accounted for by deficiencies in the physical map data rather than non-random placement of actual restriction sites. Estimates of the numbers of sites missing from the map are given, based both on the map data itself and on the site frequencies in a sample of sequenced E. coli DNA. We conclude that 5 to 15% of the mapped sites represent multiple sites in the DNA sequence.     

The nucleotide sequence of asparagine tRNA from Escherichia coli.

The nucleotide seuquence of Escherichia coli asparagine tRNA was determined to be pU-C-C-U-C-U-G-s4U-A-G-U-U-C-A-G-D-C-G-G-D-A-G-A-A-C-G-G-C-G-G-A-C-U-Q-U-U-t6A-A-phi-C-C-G-U-A-U-m G-U-C-A-C-U-G-G-T-phi-C-G-A-G-U-C-C-A-G-U-C-A-G-A-G-G-A-G-C-C-AOH. Its D-stem and D-loop have almost the same sequence as Escherichia coli aspartate tRNA.     

Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes.

The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E. coli. The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease. No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease. The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases. In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC). M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence. The Sau96I endonuclease appears to act as a monomer.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.     

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.     

The complete nucleotide sequence of the tryptophan operon of Escherichia coli.

The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism. Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level. In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E. coli together with the internal and flanking regions of regulatory information.     

The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon

The nucleotide sequence of the biotin (bio) biosynthetic operon of Escherichia coli has been determined. The 5.8-kilobase region contains the five biotin operon genes, bioA, B, F, C, and D. and an open reading frame of unknown function. The operon is negatively regulated and divergently transcribed from a control region between the bioA and bioB genes. The product of the bioA gene, 7,8-diaminopelargonic acid aminotransferase, was discovered to be related to ornithine aminotransferase. The product of the bioF gene, 7-keto-8-aminopelargonic acid synthetase, was found to be similar to 5-aminolevulinic acid synthetase.     

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.