Closer look at the Prokaryotic RNA Polymerase : Sigma factors

1
Closer look at the Prokaryotic RNA Polymerase
Sigma factors

• There are 7 sigma factors in E. coli differ in
  their affinity for the Core enzyme.
• They dont all recognize the same promoters,
  either.
• Sigma factors are themselves regulated, e.g.,
• ?32 becomes activated by heat shock (42?C).
• Displaces ?70 from the core.
• Directs the RNAP to heat-shock promoters.

2
Conserved regions of sigma factors
2.1 - Core binding 2.3 - DNA melting 2.4 -
(-10) recognition
4.1 - Activators bind 4.2 - (-35) recognition
Polymerase core has non-specific affinity for DNA
that is reduced by ? factors, thereby promoting
specific binding.
3
Figure 6.5
4
Function of s

• Provides promoter specificity
• Increases affinity of RNA Pol for promoter
• Allows transcription initiation
• Stimulates transcription initiation,
• not elongation

5
What are the functions of the other subunits of
the E. coli RNAP?
Identifying the active site.
6
Drugs that inhibit E. coli RNAP

• Rifampicin - blocks at initiation (Open Complex
  forms), rifampicin resistance mutations mapped to
  the b subunit.
• Streptolydigin- inhibits elongation stage,
  resistance mutations also map to b subunit.
• What do initiation and elongation have in common?
  Phosphodiester bond formation. Suggests ß subunit
  is key.

7
Fig. 6.33
Direct evidence for ß active site by Affinity
Labeling.
(1) Reagent in (a) resembles ATP, but it reacts
with NH2 groups when binds to active site. (2)
Then add radioactive a -32P-UTP, which is added
(i.e., polymerized) onto reagent I.
8
(3) Separate enzyme subunits by
SDS-polyacrylamide gel electrophoresis, and see
which ones are radioactive. Only the b subunit
is highly radioactive. b subunit must have
active site.
Fig. 6.33
9
E. coli RNA polymerase (redux)

• Functions of other subunits
• a - binds the UP element found upstream of very
  strong promoters (rRNA), and activators.
• b - active site of Pol, also binds nascent RNA,
  RNA-DNA hybrid, and DS DNA in front of the
  bubble.
• b also binds nascent RNA, RNA-DNA hybrid, and
  DS DNA in front of the bubble.

10
Nudler et al., 1998, Science 281, pg. 425
11
Thermus aquaticus RNAP core.
The Claw
Fig. 6.38
12
RNAP Backsliding and Editing

• If wrong nucleotide incorporated, elongation can
  become arrested.
• Backsliding now competes with elongation
• Pol backs up, extruding some of nascent RNA
• Gre proteins activate RNAP core to cleave small
  piece that has wrong nucleotide.
• Pol starts elongating again.

13
RNAP core
Square is the next NTP to be added. Green
nascent RNA that will be cleaved off Red
older RNA
14
Gene Regulation in Prokaryotes

• Regulation occurs at every level
• Gene organization (operon promotes co-expression
  of related genes)
• Transcription (repression, activation,
  attenuation)
• mRNA stability (affected by translation and the
  3 stem- loops), have degradosome
• Translation (repression, activation and
  autoregulation)
• Protein stability and other modifications
• TRANSCRIPTIONAL CONTROL MOST DOMINANT!

15
Lactose (Lac) Operon

• Diauxic growth
• Operon organization
• Negative and positive regulation
• Lac Repressor (lacI gene)
• Catabolite AP (crp gene)

16
Diauxic growth of E. coli on a mixture of lactose
glucose.
17
If E.coli presented with glucose lactose, use
mainly glucose until gone, then use lactose.
18
Lac Operon Repression
Fig. 7.3
19
Inducer Allolactose, produced by lacZ

• b - 1,6 linked

Fig. 7.4