gene regulation ii biochemistry 302 february 27,...

Gene regulation IIBiochemistry 302

February 27, 2006

Molecular basis of inhibition of RNAP by Lac repressor

CRP/DNA complex

−35 promoter site

−10 promoter site

Lewis, M. et al. (1996) Science 271:1247

−60

Lehninger Principles of Biochemistry, 4th ed., Ch 28

IPTG may serve to drive Lac repressor DNA-binding helices apart

Lewis, M. et al. (1996) Science 271:1247

no IPTG+ IPTG (note change in position of dashed transparent helices)

N-terminal subdomainof core

C-terminal subdomainof core

Hinge helix region: an allosteric switch

• no IPTG bound– Packed α-helical “hinge”– α-helices stabilized by DNA

(operator) and core contacts• w/ IPTG bound

– Rotation and translation of N-term subdomains

– Destabilization of the hinge helices

– Unpacking of hinge helix →headpiece “unfolding”

– Loss of specific DNA contacts: concerted folding and binding in reverse

– Repressor now has same low affinity for operator and non-operator sequences

Conceptual models of allosteric changes that disrupt DNA-binding

Rotation and translation

Functional groups in DNA that facilitate protein (TF) binding

Groups used for base-pair recognition are shown in red.


3 2 3223

2 3

Amino acid-base pair interactions often observed in DNA-protein binding• Amino acid side chains

most often H-bonded to bases in DNA (Asn, Gln, Glu, Asp, Lys, Arg)

• No simple amino acid base interaction code….

• ….but some restricted interactions– Asn or Gln with N6 and N7

of adenine – Arg with N7 and O6 of

guanine• DNA binding sequences

cannot be inferred from protein structure.


Lac repressor HTH motif (~20 aa): Shape, surface complementarity (fit),

and hydrophobic interactions

HTH motif is shown in red and orange. DNA is blue.

Surface rendering of HTH motif (gray) bound to DNA (blue).

Protein and DNA separated to highlight some groups interacting via hydrophobic (orange) or H-bond contact (red).


Catabolic operons: Regulation by multiple signals/ligands that target

different TFs

CRP homodimer (subunit Mr22,000) bound to DNA. cAMP“inducer” is in red. RNAP interaction domain is yellow.

Catabolite repression: Activity of lac operon is restricted when both glucose and lactose are present. E. coli would prefer to metabolize glucose directly (via glycolysis) rather than generating it from secondary sugars.


Other side of the coin: the biosynthetic trp operon• Amino acid biosynthesis consumes energy

– Advantageous to inhibit synthesis of biosynthetic enzymes when end product (amino acid) is available.

– Regulatory goal is to repress gene activity.• E. coli trp operon (in contrast to lac)

– Trp repressor is activated by ligand (Trp) binding.– Additional regulation by premature termination of

transcription (attenuation – regulatory dimmer switch involves ribosome positioning on 5′ mRNA)

• Discovered by Charles Yanofsky, common to many biosynthetic operons encoding enzymes needed for amino acid synthesis (including Trp, Leu, and His).

• Dictated by changes in RNA secondary structure• Extends the possible range of transcription rates

(moderate to high Trp levels)

Schematic of the E. coli trp operon (regulation by Trp-induced repression)

chorismic acid → Trp

Trp inducer

Dimeric HTH protein

aporepressor(when trp levels are low) Secondary mechanism of

repression: moderate to high Trp levelsFig. 26-33

Structure(s) of the trp operon mRNA leader (trpL) sequence (162 nt)

Does the 3:4 pair structure remind you of anything?


Mechanism of transcriptional attenuation

Ribosome stalling at Trp codons due to low [Trp-tRNATrp] i.e. when Trp levels are low. This allows more favored 2:3 base-pairing at the expense of 3:4 base-pairing.

Ribosome follows closely behind RNAP as transcription proceeds. The ribosome sterically hinders 2:3 base-pairing upon encountering leader peptide stop codon.

Short leader peptide has no known cellular function. Its synthesis is merely an operon regulatory device.


Another view of attenuation emphasizing importance of the ribosome

Fig. 26-36

Regulons: Network of operons with a common regulator

• Metabolism of secondary sugars– Lactose, arabinose, and

galactose– CRP-cAMP-dependent

• Heat-shock gene system– Replacement of σ70

specificity factor by σ32

– RNA polymerase directed to different set of heat-shock gene promoters

• SOS response to DNA damage– LexA repressor– RecA protein (unique role)

σ70

σ32


Induction of SOS response in E. coli(LexA-dependent regulon)

• Cellular response to extensive DNA damage

• Induced genes mostly involved in DNA repair

• Mechanism: Proteolytic inactivation of LexArepressor– RecA/ssDNA-dependent– Interaction of ssDNA-bound

RecA stimulates intrinsic protease activity of LexA.

– LexA inactivates itself by catalyzing its own cleavage at a specific Arg-Gly bond in the middle of the protein.


gene regulation ii biochemistry 302 february 27,...

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