E coli rna polymerase redux
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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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E. coli RNA polymerase (redux)

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E coli rna polymerase redux

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.


E coli rna polymerase redux

Thermus aquaticus RNAP core.

“The Claw”

From Fig 6.35


E coli rna polymerase redux

T. Aquaticus Holoenzyme

Similar to Fig. 6.37


E coli rna polymerase redux

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


Fig 6 40

Fig. 6.40

DNA used to form the RF Complex

Locks the enzyme into the open promoter complex.


E coli rna polymerase redux

Fig. 6.41a, RF Complex


Figure 6 41b rf with removed

Figure 6.41b, RF with  removed


Fig 6 43b rf model with removed

Fig 6.43bRF Model with  removed.


Rnap backsliding and editing

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.


E coli rna polymerase redux

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

Gene Regulation in Prokaryotes

Regulation occurs at every level:

  • Gene organization (operon co-expression)

  • 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 DOMINANT!


Lactose lac operon

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)


E coli rna polymerase redux

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.


E coli rna polymerase redux

CH2OH

CH2OH

HO

O

O

OH

OH

O

OH

OH

HO

galactose

glucose

lactose

Lactose Structure & Metabolism


Figure 7 3

Lac Operon: Repression

Figure 7.3


Inducer allolactose produced by lacz

Inducer : Allolactose, produced by lacZ

b - 1,6 linked

Fig. 7.4

A side reaction of lacZ.


E coli rna polymerase redux

1965 Nobel Prize in Physiology or Medicine (for their work on the lac operon and bacterial genetics)

J. Monod F. Jacob A. Lwoff


E coli rna polymerase redux

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.

Figure 7.6

IPTG = synthetic inducer of lac operon.


Figure 7 7

Figure 7.7


E coli rna polymerase redux

There are really 3 operator regions for the Lac Operon.

CAP – activator protein

RNAP – RNA polymerase

Fig 7.10


E coli rna polymerase redux

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).


E coli rna polymerase redux

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


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