Footprinting DNAProtein Interactions Powerful and fairly rapid methods

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Footprinting DNA-Protein Interactions • Powerful and fairly rapid methods for mapping where and how

Footprinting DNA-Protein Interactions • Powerful and fairly rapid methods for mapping where and how proteins bind tightly to DNA • 2 ways: 1. DNAse I footprinting 2. DMS footprinting

DNAse I Footprinting 1. Prepare end-labeled DNA. 2. Bind protein. 3. Mild digestion with

DNAse I Footprinting 1. Prepare end-labeled DNA. 2. Bind protein. 3. Mild digestion with DNAse I (randomly cleaves DS DNA on each strand) 4. Separate DNA fragments on denaturing acrylamide gels. Fig. 5. 37 a

Fig. 5. 37 b Sample of a DNase I footprinting gel. Footprint Samples in

Fig. 5. 37 b Sample of a DNase I footprinting gel. Footprint Samples in lanes 2 -4 had increasing amounts of the DNA-binding protein (lambda protein c. II); lane 1 had none.

Dimethylsulfate (DMS) Footprinting 1. End-label DNA fragment. 2. Bind protein. 3. Treat with DMS,

Dimethylsulfate (DMS) Footprinting 1. End-label DNA fragment. 2. Bind protein. 3. Treat with DMS, methylates purines. 4. Partially cleave DNA at the methylated bases. 5. Separate fragments on gel. Fig. 5. 38 a

Example of DMS footprinting. Lanes 1 and 4 had no protein Lanes 2 and

Example of DMS footprinting. Lanes 1 and 4 had no protein Lanes 2 and 3 had 2 different amounts of protein. Protein binding protects most purines from modification by DMS, but it can stimulate modification of those in regions where the helix is distorted or partially melted (indicated by *). Fig. 5. 38 b

Positive Control of Lac Operon • Catabolite Repression hypothesis – predicted that glucose would

Positive Control of Lac Operon • Catabolite Repression hypothesis – predicted that glucose would inhibit synthesis of other sugar metabolizing pathway enzymes (e. g. , lactose pathway) • Partially right, its lack of activation instead of true repression – Cells respond to high glucose with lowered levels of c. AMP and vice-versa – c. AMP activates Lac operon via CAP

cyclic 5’-3’ phosphodiester in c. AMP glucose c. AMP - Stimulates Lac operon (lac.

cyclic 5’-3’ phosphodiester in c. AMP glucose c. AMP - Stimulates Lac operon (lac. Z production) as the co-activator for the CAP protein CRP bends -->

CAP (catabolite activator protein), a. k. a. crp (c. AMP receptor protein) gene •

CAP (catabolite activator protein), a. k. a. crp (c. AMP receptor protein) gene • • CAP only active bound to c. AMP CAP-c. AMP stimulates transcription by promoting formation of closed complex: RNAP + Pro ↔ RPc → RPo Kb k 2 (RPc = Closed complex) (RPo = Open complex) Kb – equilibrium binding constant formation of RPc k 2 – rate constant formation of RPo • CAP-c. AMP increases Kb

Lac Control Region • CAP binds just upstream of promoter • L 1 deletion

Lac Control Region • CAP binds just upstream of promoter • L 1 deletion mutant has constitutively low expression Fig. 7. 16

CAP-c. AMP dimer interacts with the CTD of the subunits of the RNAP Core

CAP-c. AMP dimer interacts with the CTD of the subunits of the RNAP Core CAP-c. AMP is a dimer that binds to a short sequence (~20 bp) with dyad symmetry (activator site) αCTD binds DNA too CTD NTD - carboxy-terminal domain amino-terminal domain Fig. 7. 19

CAP-c. AMP- CTD and CAP-c. AMP-DNA complexes: CAP-c. AMP bends the activator DNA Fig

CAP-c. AMP- CTD and CAP-c. AMP-DNA complexes: CAP-c. AMP bends the activator DNA Fig 7. 17

Why does the Lac Operon need an activator? Not a very good core promoter:

Why does the Lac Operon need an activator? Not a very good core promoter: -35 -10 TTTACAC -------- TATGTT (Lac) -35 -10 TTGACAT -------- TATAAT (consensus) CAP stimulates more than 100 promoters!

Tryptophan operon: Regulation by attenuation • • Genes for tryptophan synthesis Repressed by end-product

Tryptophan operon: Regulation by attenuation • • Genes for tryptophan synthesis Repressed by end-product of pathway, Tryptophan Repression requires Operator sequence, Aporepressor (trp. R gene product) & Corepressor (Tryptophan) - Operator is within the promoter Also controlled by attenuation in the “Leader” region of the transcript

Low [tryptophan], aporepressor doesn’t bind Operator, transcription on! High [tryptophan], repressor (aporep. + tryp.

Low [tryptophan], aporepressor doesn’t bind Operator, transcription on! High [tryptophan], repressor (aporep. + tryp. ) binds operator, represses transcription! Attenuation-->

Transcription stops in the leader-attenuator “L” region when the [tryptophan] is elevated.

Transcription stops in the leader-attenuator “L” region when the [tryptophan] is elevated.

The trp Leader peptide (14 aa) has two key tryptophan codons. The ribosome stalls

The trp Leader peptide (14 aa) has two key tryptophan codons. The ribosome stalls at the trp codons when [tryptophan] is too low. The stalled ribosome prevents a downstream transcription terminator (IR + U-rich sequence) from forming. Fig. 7. 31

Fig. 7. 32

Fig. 7. 32

Biological advantage: • Repression alone decreases expression 70 -fold • Repression plus attenuation decreases

Biological advantage: • Repression alone decreases expression 70 -fold • Repression plus attenuation decreases expression 700 -fold How is translation of the downstream genes achieved with the leader peptide there to stop the ribosomes?