Mechanism of transcriptional activation by FIS: role of core promoter structure and DNA topology.
The Escherichia coli DNA architectural protein FIS activates transcription from stable RNA promoters on entry into exponential growth and also reduces the level of negative supercoiling. Here we show that such a reduction decreases the activity of the tyrT promoter but that activation by FIS rescues tyrT transcription at non-optimal superhelical densities. Additionally we show that three different "up" mutations in the tyrT core promoter either abolish or reduce the dependence of tyrT transcription on both high negative superhelicity and FIS in vivo and infer that the specific sequence organisation of the core promoter couples the control of transcription initiation by negative superhelicity and FIS. In vitro all the mutations potentiate FIS-independent untwisting of the -10 region while at the wild-type promoter FIS facilitates this step. We propose that this untwisting is a crucial limiting step in the initiation of tyrT RNA synthesis. The tyrT core promoter structure is thus optimised to combine high transcriptional activity with acute sensitivity to at least three major independent regulatory inputs: negative superhelicity, FIS and ppGpp.
Auner
Helge
H
Institut für Genetik und Mikrobiologie, LMU, München, Maria-Ward-Str 1a, 80638, München, Germany.
Buckle
Malcolm
M
Deufel
Annette
A
Kutateladze
Tamara
T
Lazarus
Linda
L
Mavathur
Ramesh
R
Muskhelishvili
Georgi
G
Pemberton
Iain
I
Schneider
Robert
R
Travers
Andrew
A
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004278:DNA, Superhelical; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000378:metabolism
D004278:DNA, Superhelical; D011196:Potassium Permanganate; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase