BCH 110C Lectures 9 and 10

In what 2 ways can the regulation of the DNA binding activity of an activator protein by a small molecule ligand turn on the transcription of a bacterial gene?

1. Addition of ligand turns gene off by removing the activator. 2. Removal of ligand turns gene off by removing the activator.

In what 2 ways can the regulation of the DNA binding activity of a repressor by a small molecule ligand turn off the transcription of a bacterial gene?

1. Addition of ligand turns gene on by removing the repressor. 2. Removal of ligand turns gene on by removing the repressor.

Describe how you can tell where a DNA binding protein binds on a DNA region using DNase I footprinting.

1. DNA fragment is labeled at one end of the strand. 2. In control, no protein is added. 3. In experimental, sequence specific DNA binding protein is added. 4. Low [DNase 1] is added. It is an endonuclease and cuts DNA randomly. 5. Bound protein will block DNase 1 activity. 6. DNA is purified (DNase killed) and its chromatographed on a denaturing gel. 7. Gel is dried and autoradiographed. Where the sequence-specific protein is bound, an empty space (the footprint) will appear between the bands produced by cleaved DNA.

How do sequence specific DNA binding proteins contact DNA to recognize specific sequences?

1. The shape of the protein fits DNA 2. Multiple interactions between + amino acids side chains of proteins and - DNA phosphate oxygen. 3. Contact involves 20 ionic, H-bond, or hydrophobic interactions in major groove or minor groove of DNA 4. Protein alpha-helices and Beta-sheets interact with DNA major groove

Repressor

A protein that decreases transcription on a promoter by physically blocking RNA Polymerase access to the promoter

Describe the CAP transcription factor subunit structure and its role in transcriptional regulation of the lactose operon. Where does it bind in the promoter and what is this site called?

Because the Lac promoter is weak, an activator (CAP) is needed to make it more efficient. CAP needs cAMP, which induces a conformational change that enables it to bind to its DNA site (CAP binding site). It promotes the recruitment of RNA Polymerase, and stabilizes binding of RNA Pol to promoter.

Silencer

DNA binding site that binds proteins that inhibits transcription at distant promoters.

Enhancer

DNA binding site that binds proteins that stimulates transcription at distant promoters.

Describe the subunit structure of the lac repressor

Each subunit can bind one molecule of allolactose. Lac repressor tetramer can bind to +11 operator and other secondary operators in the lac promoter region

Describe the effect of the distance of a regulatory protein bound to an enhancer sequence from the transcriptional initiation site on the rate of initiation of RNA polymerase at the promoter.

Enhancers and silencers can affect transcription independently of distance. Graph shows that effectiveness is highest at 500 nucleotide pairs. Too close= can't come into contact due to relatively stiff DNA.

What 3 genes (coding regions) make up the lac operon? What do they do?

LacZ gene: encodes for Beta-galactosidase, which is an enzymes that cleaves lactose LacY gene: encodes for Lactose Permease, which transports glucose into the cell LacA gene: encodes for Transacetylase, which detoxifies and acetylates pyranosides produced by B-galactosidase activity; makes it so pyranosides can't reenter cell due to aceylation

Describe the role of the NtrC protein and σ54 in the regulation of transcription initiation on the glutamine synthetase (GlnA) promoter

NtrC induces DNA looping and "open complex" formation : activates GlnA promoter 1. NtrC binds enhancer sequence upstream of GlnA promoter 2. sigma factor 54 holoenzyme of RNA polymerase binds the promoter. 3. DNA looping brings NtrC into contact with sigma factor 54 holoenzyme. 4. ATP hydrolysis drives transition from closed to open by sigma factor 54 5. Polymerase initiates.

Lac Repressor

Product of the i gene; binds to the operator tightly; lac repressor + inducer do not to DNA; competition for promoter by Lac Repressor and RNA polymerase

Describe the role of the lac repressor in transcriptional regulation of the lactose operon. Include in this answer a discussion of the structure, DNA binding, and the DNA binding sites of the repressor.

The Lac repressor, when allolactose is absent, binds its specific DNA binding site (operator) tightly. No lactose: repressor binds the operator and prevents transcription of Lac mRNA by preventing RNA Pol from binding Lactose Present: Allolactose binds repressor, which can't bind to the operator, allowing RNA Polymerase to bind the promoter.

Operator

a segment of DNA that a repressor can bind to

Describe the molecular mechanisms for the regulation of the lactose (lac) operon by lactose and glucose. Use figure(s), but also briefly explain them using sentences. As part of your answer you should identify the genes in the operon, and discuss what happens when: Glucose is absent and lactose is absent; Glucose is absent and lactose is present; Glucose is present and lactose is absent. Glucose is present and lactose is present. Be sure to include the regulatory proteins and small molecules

a) Glucose is absent and lactose is absent: Operon is off. Lac repressor binds because allolactose is (inducer) absent. CAP binds because [cAMP] is high. b) Glucose is absent and lactose is present: Operon is on. Lac repressor does not bind DNA because allolactose is (inducer) present. CAP binds because [cAMP] is high. c) Glucose is present and lactose is absent: Operon is off. Lac repressor binds because allolactose is (inducer) absent. CAP does not bind because [cAMP] is low. d) Glucose is present and lactose is present: Operon is off. Lac repressor does not bind DNA because allolactose is (inducer) present. CAP does not bind because [cAMP] is low.

Catabolite gene Activator Protein (CAP)

activator of the Lac promoter, allows it to bind RNA polymerase more tightly to the promoter; needs cAMP in order ti bind its DNA site

Describe the role of cAMP in transcriptional regulation of the lactose operon.

cAMP is a positive effector. It binds to CAP and causes a conformation change that enables it to bind to its specific DNA. It is essential for translation initiation.

How can some transcription regulatory proteins regulate transcriptional initiation when they are bound to DNA sequences which are remote from the site of transcription initiation? Give a specific example.

example: NtrC can activate transcription from a remote DNA binding site (enhancer) by inducing DNA looping NtrC is a sequence specific DNA binding transcription factor. It has ATPase activity that helps melting of DNA by sigma factor 54 over -12 promoter region for transcription initiation.

RNA Polymerase Core Enzyme and Holoenzyme

form of eukaryotic RNA polymerase that is recruited to the promoters of protein-encoding genes (mRNA)

Positive Effector

ligands that increase gene expression by selectively binding to a protein (cAMP to CAP)

Inducer

molecule that regulates gene expression; disables repressor (ex. allolactose)

Sigma Factor 54

promoter consensus sequence on RNA Polymerase found at -24 & -12 regions; used for transcription of nitrogen metabolism genes; requires activators with ATPase activity that bind DNA sequences (enhancers); enhancers are activators (help RNA polymerase bind to the promoter)

Beta-galactosidase

synthesized by lacZ gene; enzyme that cleaves lactose into glucose and galactose