Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL.
The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.
Brown
K L
KL
School of Biological Sciences, University of East Anglia, University Plain, Norwich, UK.
Wood
S
S
Buttner
M J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D018834:Chaperonin 60; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006360:Heat-Shock Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C071941:HrdB protein, Streptomyces; D012321:DNA-Directed RNA Polymerases