E. coli RNA polymerase (redux) Functions of other subunits: α - binds the UP element upstream of...
-
Upload
morris-beasley -
Category
Documents
-
view
218 -
download
0
Transcript of E. coli RNA polymerase (redux) Functions of other subunits: α - binds the UP element upstream of...
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.
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.
Square is the next NTP to be added.
Green – nascent RNA that will be cleaved off
Red – “older RNA”
RNAP core
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.
OOH
CH2OH
O
OH
CH2OH
OHO
HO
OH
OH
galactose glucose
lactose
lactose
Glucose Galactose
epimerase
glycolysis
-galactosidase
Lactose Structure & Metabolism
J. Monod F. Jacob A. Lwoff
1965 Nobel Prize in Physiology or Medicine (for their work on the lac operon and bacterial genetics)
Example: lacI + DNAo ↔ lacI-DNAo
lacI – lac repressorDNAo – lac operator DNA
Kd = [lacI] [DNAo] ∕ [lacI-DNAo]Kd = equilibrium dissociation constant
1 x 10-8 to 10-12 M = high affinity
Equilibrium DNA – Protein Binding
There are really 3 operator regions for the Lac Operon.
CAP – activator proteinRNAP – RNA polymerase
Fig 7.10
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).
Operators work cooperatively (synergistically).