Gene cloning and characterization of a novel extracellular ribonuclease of Bacillus subtilis.
An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA-containing agar medium. An open reading frame encoding 289 amino acids was identified within the cloned fragment. The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the E sigma A and/or E sigma E RNA polymerases. The production of the nuclease by the B. subtilis transformants greatly depends on the liquid medium used. SDS/PAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa, and the NH2-terminal amino acid sequence analysis suggested that the NH2-terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion. The nuclease was uniquely characterized as a Mg(2+)-activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5'-terminal phosphate. The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences.
Nakamura
A
A
Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
Koide
Y
Y
Miyazaki
H
H
Kitamura
A
A
Masaki
H
H
Beppu
T
T
Uozumi
T
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D002413:Cations, Divalent; D004269:DNA, Bacterial; D004722:Endoribonucleases; D012260:Ribonucleases; D015246:Deoxyribonuclease EcoRI; C077268:nuclease Bsn; D008274:Magnesium