Dataset Information

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

ABSTRACT: A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.

SUBMITTER: Itaya M

PROVIDER: S-EPMC328632 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

Itaya M M Kondo K K

Nucleic acids research 19910801 16

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophi ...[more]

PMID: 1653414

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Project description:Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To further characterize functional significance, we successfully mapped 113 acetylation sites on their 54 authentic and 59 homologous protein structures. The acetylation in the majority of proteins occurs in ordered structures and the sites were situated near the negatively charged glutamic acid residues. In addition, 59 of 103 acetylations were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylations. Finally, we further summarized 22 critical acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and protein-protein interaction. The structural information of 113 acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins. Data processing, bioinformatics: MS and MS/MS spectral data were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The carbamidomethylation of cysteine was selected as a fixed modification. The oxidation of methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified.

Dataset Information

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

Publications

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

Similar Datasets

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