Core RNA polymerase and promoter DNA interactions of purified domains of sigma N: bipartite functions.
The sigma N class of sigma factors confer upon RNA polymerase the requirement for enhancer-binding activator proteins. The sigma-N (sigma N) protein of Klebsiella pneumoniae was analysed by the assay of purified peptides comprising domains or regions of sigma N defined by proteolysis or by homology alignment, respectively. The NH2-terminal Region I is required for the correct interaction of holoenzyme with the promoter, and promoter complexes forming with a truncated sigma N lacking Region I are not activatable. The complexes lack the DNA structure believed to represent nucleated strand separation but still make close contacts with this promoter part. Determinants of specific DNA recognition by sigma N were shown to reside in a C-terminal 16 kDa peptide, and core RNA polymerase binding determinants in an adjacent peptide. The latter contacts and appears to pack against the DNA-binding domain. Thus the DNA-binding and core-binding domains are bipartite in function, consistent with core functioning as an allosteric effector of the sigma DNA-binding activity. The DNA-binding and core-binding domains together include Region III of sigma N. Although not the primary determinant of core or DNA recognition, the acidic Region II of sigma N influenced both activities. Regions I and II in combination with core RNA polymerase thus appear to control the activity of C-terminal DNA contacting surfaces to allow formation of a closed promoter complex that is susceptible to activation.
Cannon
W
W
Nitrogen Fixation Laboratory, University of Sussex, Falmer, Brighton, England.
Missailidis
S
S
Smith
C
C
Cottier
A
A
Austin
S
S
Moore
M
M
Buck
M
M
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000502:physiology; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000502:physiology
D004269:DNA, Bacterial; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases