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“Hybridization competition”
Sigma (σ) as a Specificity Factor
Sigma (σ) as a Specificity Factor
Green: 1min
Red: 5 min
Blue: 17 min after infection
with T4 phage
The Core Enzyme Transcribes
Both DNA Strands
ting for asymmetric or symmetric transcription in vitro
g hybridization and checking subsequent RNase resist
Core enzyme
Holoenzyme
Sigma (σ) as a Specificity Factor
hy was core RNA polymerase still capable of transcrib
nicked DNA, but not intact DNA?
Sigma (σ) as a Specificity Factor
hy was core RNA polymerase still capable of transcrib
nicked DNA, but not intact DNA?
+ An excess of
unlabeled DNA
Binding of RNA Polymerase to Promoters
Nitrocellulose filter-binding studies
performed by Hinkle and Chamberlin
+ An excess of
unlabeled DNA
Binding of RNA Polymerase to Promoters
Consensus sequences
UP Core
element promoter
UP Core
Fis sites element promoter
- Many oligonucleotides/polymerase
- Abortive transcripts up to 9 or 10 nt
in size
Transcription Initiation
“Promoter clearance”:
The transcript becomes long
enough to form a stable hybrid
with the template strand
(pyrophosphate)
: Fluorescence
acceptor
: Fluorescence
donor
FRET Analysis of σ-core Association After
Promoter Clearance
Dimethyl sulfate
(DMS)
- Melted region of ~ 12 bp
- The melted region is just at the place
where RNA polymerase begins transcribing.
Local DNA Melting at the Promoter
Howard Gamper and John Hearst (1982):
“Transcription bubble”:
Moves with the polymerase
as it transcribes the DNA
Structure and Function of σ