Lecture 33, 11/24; Chapter 17 Regulation of Gene expression

Chapter 17 Regulation of Gene expression in Eukaryotes

Positive induction and catobolite regulation in yeast.
What are we looking at: The regulation of genes involved in galactose breakdown in yeast.

• In the absence of galactose genes are off.

• Presence of galactose the genes are expressed. (energy is conserved)
• Galactose genes are only expressed when glucose levels are low - catoblite repression.
• A mutation in the GAL 4 gene prevents the activation of the galactose genes even under appropriate conditions.
  

(6:00) What does GAL 4 do?

• GAL 4 functions as an activator.
• When galactose is present GAL 4 turns on the galactose genes.  So . . . when you mutate it and it can't function you will never see activation.
  

Catabolite repression - requires an enzyme.  This is opposed to an E. coli in the lac operon where catabolyte repression is based on cyclic amp levels.

(8:30) We're going to look at the regulation of GAL 1 to GAL 10

• upstream of both genes - you have a DNA sequence called UASg (upstream activator sequence).  It is a 170 BP sequence that acts as an enhancer.
• within the UAS there are 4 binding sites for the GAL 4 protein (the activator protein).  These sites are always bound by GAL 4 protein.  The activator is always present HOWEVER, the genes are not always expressed.
• (12:00) How is it that they are not expressed?
• A second protein called GAL 80 protein can physically interact with the GAL 4 protein. (form a protein protein complex)
  
• GAL 80 protein blocks the portion of the GAL 4 protein which activates transcription.
  
• GAL 80 protein is always bound to GAL 4 protein
• How this works: To induce . . . galactose, bound to a phosphate group, binds to the GAL 4/GAL 80 complex.  In doing so alters the interaction between GAL 4 and GAL 80 in such a way as to expose the region of GAL 4 that activates transcription. 
  
• In the presence of glucose a protein kinase (an enzyme which phosphorilates things) goes to the promoter to repress transcription.

(18:40) Post transcriptional regulation of gene expression.  (side note review: Eukaryotic gene regulation happens at different points)

• often these processes occur during mRNA modification (modification being: splicing, 5' capping and polyadenylation).  You have this period of time (post transcription, pre translation) where you alter structure of mRNA which opens a window to allow for altering expression of a gene.  ONe of the major forms of this is alternative splicing.
  
• Alternative splicing: splicing is the process of removing introns from mRNA molecules.  If you change the splicing pattern (don't remove an intron) you will change the protein that an mRNA can make.
• What this does: Alternative splicing allows the proteone (the number of proteins in a cell) to be greater than the genome (number of genes in the cell).  It is proposed that this is a common phenomenon in vertebrates.  IN fact 30 to 60% of all human genes are alternatively spliced. 
  
• While the genome has 25-30,000 genes, the proteon has 100,000's of proteins. 
  

(25:10) How alternative splicing impacts cell function.
Cochlear hair cells and hearing. 

• Broadly: Hair cells beat to allow the air to hear different frequencies.  This is controlled by the alternative splicing of the mRNA.
  
• SLO gene contains 8 different alternative splicing sites.  This gene can be spliced into 500 different mRNA's.
• Each different protein is responsible for a different frequency.  Essentially you have used alternative splicing to create proteins to function for a broad cellular function.
  

(30:40) Use of a regulatory RNA to control gene expression.

• There is a short, 21 nucleotide, RNA that represses the translation of certain RNA molecules.  Similar RNA's have been found in the nucleus - which specifically bind and repress genes at the level of DNA. Ex. - RNAi and PTGS
  
• RNAi (RNA interference) in animal cells. Post transcriptional gene silencing (PTGS) in plants. 
  
• The process begins with the creation of a double stranded RNA 70 BP long.  It is then processed by a protein called dicer protein.  The dicer protein cleaves the RNA to 21 nucleotides (the result is referred to as an siRNA or short interfering RNA)  siRNA Binds to a protein called "RNA Induced Silencing Complex".  RNA binds to complimentary mRNA molecules and turns off their translation.  
  
• (36:20) miRNA or micro RNA - 19-24 nucleotides. Binds to a 3' untranslated region of an mRNA to turn it off.

Turning off the translation is valuable as it is critical to development and helps the cell ward off viruses.  RNA interference (discovered within past 5-10 years) is important and is being thought of as a potential theraputic agent.  Introduce RNA complimentary to a mutated gene and it will seek out hte bad RNA's to turn them off.