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.

From Fig 6.35

Thermus aquaticus RNAP core. “The Claw”

T. Aquaticus HoloenzymeSimilar to Fig. 6.37

RNAP binds/protects DNA from bp -44 to +3

Fig. 6.40

DNA used to form the RF Complex

Locks the enzyme into the open promoter complex.

Fig. 6.41a, RF Complex

Figure 6.41b, RF with removed

Fig 6.43bRF 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.

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

Figure 7.3

Lac Operon: Repression

Inducer : Allolactose, produced by lacZ

- 1,6 linkedFig. 7.4 A side reaction of lacZ.

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

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 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).

Structural basis for cooperativity of operators: Lac repressor can bind 2 operator sequences.

LacI Tetramer(2 dimers held together at the bottom)

DNA

Fig 7.12a