Identification of rpoBC genes encoding for beta and beta' subunits of RNA polymerase in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12.
RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was sigma(70) containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the beta and beta' subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni(2+) affinity column. In vitro reconstitution using all proteins for the holoenzyme (alpha, beta, beta', sigma(70)) was carried out and the activity of the recombinant RNA polymerase was detected.
Kawano
Hiroaki
H
Extremobiosphere Research Center (XBR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan. kawanoh@jamstec.go.jp
Nakasone
Kaoru
K
Abe
Fumiyoshi
F
Kato
Chiaki
C
Yoshida
Yasuhiko
Y
Usami
Ron
R
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008386:Marine Biology; D008969:Molecular Sequence Data; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology*
D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases