FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.
We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.
Pemberton
Iain K
IK
Enzymologie et Cinétique Structurale, UMR 8532 du CNRS, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan, France.
Muskhelishvili
Georgi
G
Travers
Andrew A
AA
Buckle
Malcolm
M
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D007700:Kinetics; D007834:Lasers; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; C440502:integration host factor, E coli; D012321:DNA-Directed RNA Polymerases