Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase.
RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.
López de Saro
F J
FJ
Section of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
Woody
A Y
AY
Helmann
J D
JD
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D011099:Polyglutamic Acid / Q000378:metabolism; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012808:Sigma Factor; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012808:Sigma Factor; D014157:Transcription Factors; C063202:sporulation-specific sigma factors; D011099:Polyglutamic Acid; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases