Lecture 32, 11/21; Steps to Transcription in Eukaryotes

Transcription in Eukaryotes requires multiple steps

• It begins with chromatin remodeling. Within the structure of the chromosome we have regions of both euchromatin and heterochromatin (these regions are highly condensed and genes in these regions are not expressed). Chromatin remodeling primarily occurs to regions of euchromatin.
• Broadly: Chromatin remodeling uses a series of proteins to weaken the interaction between the histones and the DNA.
• All of these proteins have ATPase activity - they all convert ATP --> ADP (requires energy).
  
• (4:20) One such protein is the SWI/SWF complex.
• a complex of 11 subunit proteins. One of the subunits binds DNA. Another of the subunits is the ATPase subunit.

(5:35) How chromatin remodeling occurs

• Modify the interaction between DNA and Histones. This causes the Histone to slide down the DNA. (REMEMBER: We have DNA wrapped around the histone protein, that DNA that is interacting with the histone protein will be very inaccessbile.) As the histone moves the DNA that was attached to the histone, becomes exposed.
  
• The protein complexes (like the SWI/SWF complex) function to physically pull the DNA away from the histone, thus allowing access.
  

(9:10) Another way to accomplish chromatin remodeling.

• Catalyzed by histone acetyl transferase (HAT). These enzymes can add acetyl groups to the histones. Weaken the ineraction between the histone and DNA.
• When transcription is finished histone deacetylase (HDAC). They remove the acetyl groups and restore normal chromatin structure.
• Typically remodeling process starts just before the promoter for a gene and ends at the end of a gene. The portion that is going to be copied is the only thing that is opened up - to do this an insulator element is used.
  
• Insulator element binds to proteins to prevent the spread of remodeling.

(15:05) After remodeling the process moves on to the Assembly of the basal transcription complex

• Eukaryotes have multiple RNA Polymerases that transcribe different things
• RNA POL I is responsible for the transcription for rRNA.
  
• RNA POL II copies mRNA (mRNA is a major portion of the cell) and snRNA (snRNA does splicing).
  
• RNA POL III transcribes tRNA and 5SrRNA.
• each polymerase recognizes different promoter sequences.
  

(20:05) How RNA POL II initiates transcription.

• A number of proteins will come together to form a pre-initiation complex on which RNA POL II lands. The pre-initiation complex is recognized by the RNA POL II as a place to land and "act". What is involved in making the pre-initiation complex.
  
• TFIID (TF=transcription factor, II=RNA POL II, D= just a differentiating letter)
• Recognizes and binds the TATA box. A number of other proteins bind TFIID (there are many but they are not important).
  
• Ultimately RNA POL II binds this complex. After binding, RNA POL II leaves the TATA box and starts transcription at the basal level. Enhancers or silencers can alter the level of transcription.
  
• Activator proteins can enhance transcription 100 fold. (Whatever the basal level is, multiply by 100). The activator protein binds to enhancer DNA sequences to accomplish this.
  

(26:00) What does an activator protein look like? (It has two regions or domains)

• DNA binding domain - binds to DNA. Specifically an enhancer sequence.
• trans-activating domain: 30 -100 amino acids. It interacts with other transcription factors or RNA POL. If functions to increase the level of binding by RNA POL. In order to stimulate transcription you need to have RNA POL bind more frequently.

(30:00) SUMMARY
To start the process we do chromatin remodeling and open up the DNA so that it is accessible to RNA POL. After we accomplish that we assemble our complex then bind RNA POL, and in order to enhance transcription we utilize enhancer proteins.

Genetics Lecture 28, 11/10; Chap 16: Regulation of Gene Expression, Lac operon

Test Friday 11/16

Chap 16: Regulation of Gene expression in Prokaryotes

• Gene Expression: Transcription - process of converting DNA to RNA
• The cell regulates transcription and can turn on or off the transcription of a gene. It doesn't want to make something it won't use. (sensitive to how they spend energy)
  
• In prokaryotes genes are typically found by themselves, a single gene OR as operons.
  
• Operons - a group of genes which are transcribed as a single unit.
• Typically the genes found in an operon are all involved in some process together (this way they can all be regulated together)
  
• Promoter - upstream of a gene (closer to 5 prime end) site where RNA POL binds to start transcription.
• In prokaryotes there is a sigma factor - a small protein that assists RNA POL in recognizing and binding a promoter.
• After that RNA POL begins copying DNA into RNA and continues until it reaches the Terminator (which is at the end of the gene or operon) and signals the end of transcription. No more than that is copied - don't want to waste energy.
  

(8:20) How do we regulate the above process of transcription. The study of gene regulation in prokaryotes is EXTENSIVE.

• 1900 it was recognized that cells fail to produce the enzymes for lactose metabolism when lactose is absent. Gave rise to the idea that gene expression is adaptive.
  
• Constituitive expression - a gene that is always expressed at a relatively high level.

(11:20) Two types of Expression:

• Positive Regulation - the turning on of the expression of a gene. (Gene is off and then you do something that induces the expression of that gene)
  
• Negative Regulation - the gene is being expressed until you turn it off. (ex. Tryptophan)
  

(14:15) Example of Positive Regulation: Lac Operon

• Late 1940's - Jacob + Monod (links to Wikipedia article): laid the groundwork for all the understanding of gene regulation.
  
• Based on the idea that in a prokaryote the enzymes for lactose metabolism are off without lactose. Their goal was to understand this idea.
  
• lac operon: three structural genes involved in lactose metabolism
• lac Z - codes for beta galactosidase which breaks down lactose to produce glucose and galactose. You don't want to produce this if you have no lactose.
  
• lac Y - produces an enzyme called permase which is responsible for facilitating the entry of lactose into the cell.
  
• lac A - codes for transacetylase. It is believed that it helps to breakdown some of the toxic byproducts of lactose metabolism.
• cis-acting element - a DNA sequence that is bound and acted upon to allow the regulation of a genes expression (turns it on or off).
  
• trans acting factor - a molecule, often a protein, that binds a cis-acting element to regulate the expression of the gene.
• (24:00) Gratuitous inducer (found by Jacob and Monod) - a molecule which mimics the activity of a molecule which normally activates a system.
  
• Lactose induces the expression of the lac operon. The inducer can fill the role of lactose.
• IPTG (part of the gratuitous inducer) - when added to the system turned on the expression of the lac operon. This allowed them to find constitutive mutants (mutants which always express the lac operon).
• Lac I - gene upstream of the lac operon (not part of the operon). Lac I is repressor gene that produces a protein which bound and repressed the lac operon. When you mutate the lac I gene, cells consituitively express the lac operon. (Trans acting factor - something that is produced that acts upon a cis-acting element) It no longer represses the expression of the lac operon.
  
• Lac O - mutation in the operator sequence. Cis acting element. DNA sequence bound by Lac I to prevent transcription of the lac operon. (this is a constituitive mutant)
• Finding the lac I and lac O mutants allowed them to develop a hypothesis as to how the lac operon worked
  

(33:10) How the regulation of the lac operon works in the absence of sugar.

• The lac I gene produces the Lac I protein. The repressor protein binds to lac O (operator sequence)
• When this happens RNA POL binds the promoter. Repressor protein blocks RNA POL from copying the lac operon, therefore, operon repressed.

How the regulation of the lac operon works in the presence of lactose

• lac I gene produces the Lac I repressor protein. Lactose binds to the Lac I repressor protein. This triggers a confirmation change in Lac I.
  
• Because of this Lac I cannot bind the operator. SO when this happens RNA POL can bind the promoter and copy the lac operon. The result is that the lac operon is induced.