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.