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.
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Thermus aquaticus RNAP core.
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
Locks the enzyme into the open promoter complex.
Fig. 6.41a, RF Complex
Square is the next NTP to be added.
Green – nascent RNA that will be cleaved off
Red – “older RNA”
Regulation occurs at every level:
TRANSCRIPTIONAL CONTROL DOMINANT!
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
Lac Operon: Repression
b - 1,6 linked
A side reaction of lacZ.
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
Lac repressor binding to lac operator.
IPTG = synthetic inducer of lac operon.
There are really 3 operator regions for the Lac Operon.
CAP – activator protein
RNAP – RNA polymerase
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.
(2 dimers held together at the bottom)