Regulation Of Gene Expression

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Levels of Gene Expression Control

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1.) Transcriptional Control (Nucleus)

2.) Processing Control (Nucleus)

3.) Transport Control (Cytoplasm)

4.) mRNA Degradation Control (Cytoplasm) or

4.) Translational Control (Cytoplasm)

5.) Protein Degradation Control (Cytoplasm)

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General Transcription Factors (GTF's)

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Proteins that bind to promotor and recruit RNA Polymerase II

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Activators

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Proteins that stimulate transcription initiation (by 100 fold). Attach at enhancer site of DNA. Gal4p.

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Coactivators

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Large multi protein complex, doesn't bind

directly to DNA but participates in activation by interacting with activators and GTF's. "Mediator" complex.

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Repressors

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Transcription factors that inhibit the activation of transcription initiation by activators. Gal80p

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Corepressors

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Called on by repressors, work same as

coactivators.

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HATs Enzymes

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Histone Acetyl Transferases: activators bind to their binding site and histones and call on HATs. Acetylation neutralizes the positive charge of lysine residues on histones. Slowly, the positively charged histones lose affinity for negatively charged DNA, and DNA packaging loosens up. The promotor is more accessible for activation of transcription.

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HDACs

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Histone Deacetylases: activators bind to their binding site and histones and call on HDACs to remove the added acetyl groups, restoration of tight packaging around

histones.

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ATP-dependent Nucleosome Remodeling

Complexes

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Large multiprotein complexes that remodel (loosen/provide access to) chromatin by use of ATP hydrolysis. Activator binds to its binding site and calls on complexes. May SLIDE nucleosomes, RESTRUCTURE, or TRANSFER one nucleosome from one DNA molecule to the other.

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Gene Silencing

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A gene is transcriptionally silent due to its

location.

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Silencing by DNA Methylation

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Involves DNA Methyltransferases (DNMTs) modifying cytosines to produce 5-methycytosine (5mC). 5'-CG-3' = CpG dinucleotides. Specific proteins recognize the methylation and call on HDACs to tighten the histone in that area, disabling transcription.

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Genomic Imprinting

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An epigenetic phenomenon in which the expression of certain genes is determined by whether the gene is inherited from the male or female.

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RNA Processing Control

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1.)Alternative Polyadenylation: the addition of numerous adenines to the 3' end of a gene.

2.) Alternative Splicing: depending on the use of the eventual polypeptide, the RNA is spliced in different locations, getting rid of the sections it doesn't need.

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Translational Control

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mRNAs are altered so they may stay in a state at which they aren't translated. Ex: unfertilized eggs. Proteins bind to the mRNA to protect them and inhibit their translation. Stored mRNA has a short poly-A tail as, apparently, long poly-A tails signal translation.

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microRNA in Degradation/Silencing

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mRNA is looped and Poly-A tail and 5' cap are cut off. It is sent to the cytoplasm where endonuclease Dicer is added and makes staggered cuts, resulting in 2 strands. Dicer stays and Argonaut (Ago 1) is added. Ago1 cleaves miRNA* strand. The leftover 1 mRNA strand and Ago1 and Dicer are called the RISC complex. Targets mRNAs unlike itself and inhibits translation and moves it to P body for degradation or storage.

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siRNA in Degradation/Silencing

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Long dsRNA in cytoplasm. Dicer is added and makes staggered cuts resulting in many small fragments. Ago2 is added to RNA and Dicer. Ago2 cleaves one of the two strands and a helicase unwinds and separates. The siRISC complex pairs with perfect matches and takes them to P bodies.

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mRNA Degradation

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Deadenylation-Dependent Pathway: Poly A tails are deadenylated until they are too short for PAB. Then an enzyme catalyzed "decapping" occurs to 5' cap. The naked mRNA is then degraded in the 5' to 3' direction.

Independent: Some mRNA still gets degraded without using deadenylation and we don't know how.

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Protein Degradation (Proteolysis)

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Requires addition of ubiquitin proteins (76 amino acids found ubiquitously in all eukaryotes). Then transported to proteasome which holds proteases, in which tagged proteins are degraded into short polypeptides.

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Southern Blot

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Method of determining if a specific DNA fragment or sequence is present in a sample.

PCR some probes about 200bp long w/ fluorescence. Put isolated DNA (E. coli, Chlamydia, etc) electrophoresis gel and electrify it. Place gel on a sponge in a salt water solution. A positively charged nylon sheet is placed on top of gel, then paper towels. Put nylon sheet in ziplock bag and add probes. X-ray sheet. The markers that show up mean the DNA is packaged and isn't being expressed at the time.