Regions Of Amino Acid Similarity Research Articles

11] σ Factors: Purification and DNA binding

Publisher Summary This chapter focuses on purification of σ polypeptides using affinity tags, and analysis of DNA binding in vitro . The sigma (σ) subunit of prokaryotic RNA polymerases provides specificity for transcription initiation by directing polymerase to the appropriate promoter DNA sequences. The chapter describes a large number of different σ subunits, which are related to the Escherichia coli primary σ. Members of this group are characterized by four regions of amino acid similarity. The ability of σ to bind to DNA in vitro , in the absence of the core polymerase subunits varies depending on the structure of the σ polypeptide. For example, DNA binding by σ is not observed unless the amino-terminal inhibitory domain is removed. Alternative σ factors, such as Bacillus subtilis σ K and Salmonella typhimurium σ, which lack the inhibitory domain, bind to DNA in full-length form. Another family of σ factors, related to E. coli σ, contains members that are quite dissimilar to the σ family in both structure and function. Unlike σ, full-length σ is capable of binding to DNA.

Characterization of mammalian ADP-ribosylation cycles

NAD: arginine ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety from NAD to an arginine in an acceptor protein, whereas ADP-ribosylarginine hydrolases remove ADP-ribose, regenerating free arginine and completing an ADP-ribosylation cycle. A family of four mono-ADP-ribosyltransferases was isolated and characterized from turkey erythrocytes. Transferases from rabbit and human skeletal muscle were cloned. The muscle transferases are glycosylphosphatidylinositol-anchored proteins and highly conserved across mammalian species. The rat T cell alloantigen RT6.2 has significant amino acid sequence identity to the muscle ADP-ribosyltransferase. Mammalian cells transformed with the RT6.2 coding region cDNA expressed NAD glycohydrolase activity. Sequences of RT6.2, rabbit muscle transferase and several of the bacterial toxin ADP-ribosyltransferases contain regions of amino acid similarity which, in the bacterial toxin ADP-ribosyltransferases, from the NAD-binding and active-site domains. ADP-ribosylarginine hydrolase, initially purified from turkey erythrocytes, was cloned from rat, mouse, and human brain. Deduced amino acid sequences of the rat and mouse hydrolases were 94% identical with five conserved cysteines whereas the human hydrolase sequence was 83% identical to that of the rat, with four conserved cysteines. It is unclear how an intracellular hydrolase acts in concert with a surface ADP-ribosyltransferase.

Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases.

Polymerases in general share only a few regions of amino acid similarity. One of the most conserved regions, called motif A, has the sequence DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA polymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substitutions in this highly conserved motif. The mutant DNA polymerases differed from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase replicated DNA with reduced fidelity. Another mutant DNA polymerase replicated DNA with increased accuracy, but this mutant DNA polymerase was less processive in primer extension reactions, and DNA replication required high concentrations of deoxynucleoside triphosphates. We provide evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases partition between states active for DNA replication or exonucleolytic proofreading. These studies also provide further support for the hypothesis that the accuracy of DNA replication observed for DNA polymerases and 3'-->5' exonuclease activities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247, 7116-7122).

Phi 29 DNA polymerase active site. The conserved amino acid motif “Kx3NSxYG” is involved in template-primer binding and dNTP selection

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif "Kx3NSxYG" has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.

Phi 29 DNA polymerase active site. Mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding.

Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs D-NSLYP and K--NS(L/V)YG, regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.

Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein

We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport. dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA). Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein. Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source. dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase. An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly. Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal.

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12.

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.