E. coli RNA polymerase (redux)
• Functions of other subunits:
• α - binds the UP element upstream of very strong
promoters (rRNA), and some protein activators.
• β - active site of Pol, also binds , nascent RNA,
RNA-DNA hybrid, and DS DNA in front of bubble.
• β' - also binds , nascent RNA, RNA-DNA
hybrid, and DS DNA in front of bubble.
Thermus aquaticus RNAP core.
“The Claw”
From Fig 6.35
T. Aquaticus
Holoenzyme
Similar to Fig.
6.37
RNAP binds/protects DNA from bp -44 to +3
DNA used to form the RF Complex
Fig. 6.40
Locks the enzyme into the open promoter complex.
Fig. 6.41a, RF Complex
Figure 6.41b, RF with 
removed
Fig 6.43b
RF Model with
 removed.
RNAP: Backsliding and Editing
1. If wrong nucleotide incorporated, elongation
can become arrested.
2. Backsliding now competes with elongation:
– Pol backs up, extruding some of nascent
RNA
3. Gre proteins activate RNAP core to cleave
small piece that has wrong nucleotide.
4. Pol starts elongating again.
RNAP core
Square is the next NTP
to be added.
Green – nascent RNA
that will be cleaved off
Red – “older RNA”
Gene Regulation in Prokaryotes
Regulation occurs at every level:
1. Gene organization (operon co-expression)
2. Transcription (repression, activation, attenuation)
3. mRNA stability (affected by translation and the 3’
stem-loops), have “degradosome”
4. Translation (repression, activation and
autoregulation)
5. Protein stability and other modifications
TRANSCRIPTIONAL CONTROL DOMINANT!
Lactose (Lac) Operon
• Diauxic growth (2 phases or types, which
use different substrates)
• Operon organization
• Negative and positive regulation
– Lac Repressor (lacI gene)
– Catabolite Activator Protein (crp gene)
Diauxic growth
of E. coli on a
mixture of
lactose +
glucose.
If E. coli presented
with glucose &
lactose, use mainly
glucose until gone,
then use lactose.
Lactose Structure & Metabolism
CH2OH
HO
CH2OH
O
OH
l ac t ose
-galactosidase
O OH
O
OH
OH
HO
galactose
glucose
Glucose
Galact ose
lactose
ep im erase
g ly co ly sis
Lac Operon: Repression
Figure 7.3
Inducer : Allolactose, produced by lacZ
Fig. 7.4
A side reaction of lacZ.
 - 1,6 linked
1965 Nobel Prize in Physiology or Medicine (for
their work on the lac operon and bacterial
genetics)
J. Monod
F. Jacob
A. Lwoff
Equilibrium DNA – Protein Binding
Example: lacI + DNAo ↔ lacI-DNAo
lacI – lac repressor
DNAo – lac operator DNA
Kd = [lacI] [DNAo] ∕ [lacI-DNAo]
Kd = equilibrium dissociation constant
1 x 10-8 to 10-12 M = high affinity
Figure 7.6
Lac repressor
binding to lac
operator.
IPTG = synthetic
inducer of lac
operon.
Figure 7.7
There are really 3 operator regions for the Lac Operon.
CAP – activator protein
RNAP – RNA polymerase
Fig 7.10
Operators work cooperatively (synergistically).
DNAs introduced
into E. coli genome
of a lacZ mutant
using λ phage.
LacI gene was
present in the
chromosome.
IPTG was used to
induce lacZ.
Numbers are based on the ratio of lacZ activity
in the presence and absence of inducer (IPTG).
Structural basis for cooperativity of operators:
Lac repressor can bind 2 operator sequences.
DNA
LacI Tetramer
(2 dimers held
together at the
bottom)
Fig 7.12a