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.

Lecture 31, 11/19; Chapter 17, Regulation of Gene Expression in Eukaryotes

Regulation of gene expression in eukaryotes

Gene expression in a multi-cellular organism is very different than in a prokaryote. One such difference is cellular differentiation.

-Cellular differentiation: start out with general undefined cells then turn on different genes to make a different type of cell or cell types. This process is CRITICAL. You need to turn on the correct genes at the correct times otherwise you will get death.

• ex. of cellular differentiation --> you need different things in a muscle cell then in a nerve cell.

(3:35) How the regulation of gene expression differs in Eukaryotes

• Eukaryotic cells are larger and more complex than prokaryotes. Within this, DNA is packed into chromatin with histone proteins. Chromatin remodeling is a key step in the regulation of gene expression in a eukaryote. So if you don't have your DNA in a form that is accessible to RNA POL to copy you can essentially turn off transcription by shuttin down chromatin remodiling.
  
• Eukaryotes typically have their DNA in multiple chromosomes not one chromosome (typically seen in prokaryotic organism)
  
• Because DNA is in the nucleus and the ribosomes are at the endoplasmic reticulum (ER) in the cytosol, transcription and translation are seperated spatially and temporally (occur at different times). In a prokaryote: almost as quickly transcription starts to produce RNA, ribosomes come in, bind that RNA and start to translate that RNA. In eukaryotes these processes are seperated.
• mRNA molecules are modified prior to exiting the nucleus in a eukaryote. That modification includes splicing.
  
• (8:50) Eukaryotic mRNA molecules are more stable than prokaryotic, they have a longer half-life (amount of time they exist). Partly because Prokaryotes need to rapidly respone to changing conditions. Eukarytoes don't experience this as much.
• In eukaryotes regulation can also occur at the level of translation. You have a more stable mRNA molecule but you may alter or regulate the translation of an individual mRNA molecule because it's not being produced and degrated as quickly.
  
• While all eukaryotic cells contain complete copies of their genome (all chromosomes + DNA in all cells) different cells express different subsets of genes.
• Broadly: the process of regulation in a eukaryote is a more complex process then what is seen in a prokaryote.

Differences in regulation of gene expression	Prokayote	Eukaryote
1st difference		larger and more complex
2nd difference	DNA typically in one chromosome	DNA in multiple chromosomes
3rd difference	transcription and translation happen almost simultaneously	transcription and translation are seperated spatially and temporally
4th difference		mRNA molecules are modified prior to exiting the nucleus
5th difference	Prokaryotes need to rapidly respond to changing conditions - less stable.	more stable, longer half-life
6th difference		regulation can also occur at the level of translation
7th difference		contain complete copies of their genome in each cell

(14:45) Chromosome organization in the nucleus influences gene expression

• During interphase, the DNA found in the nucleus is in a relaxed state. However, there is stil orginization to how DNA is organized within the nucleus. This organization plays a key role in the regulation of gene expression.
• Within the nucleus, each unique chromosome exists in a chromosome territory.
• The regions between the chromosome territory's are called interchromosomal domains.
• (19:45) Within the nucleus the arrangement is as follows:
• Chromosomes with small numbers's of genes have thier chromosome territory on the outside of the nuclues.
• Chromosomes with larger numbers of genes exist in chromsome territiores towared the inside of the nucleus.
• It has been proposed that the genes being actively transcribed on a chromosome will be found toward the edge of the chromosome territory. This suggests that RNA POL lives in the interchromsomal domains. The genes need to be brought close to those areas so they can be transcribed.
  
• Once we get a chromosome in position the intition of transcription begins --> two steps.
• Chromatin Remodeling
• We need to recruit a number of factors (typically proteins) that help initiate transcription.

(27:10) Transcription Initiation

• There are three common cis acting elements in Eukaryotic transcription initiation: promoters, enhancers and silencers.
• The process requires chromatin remodeling, a number of DNA sequences and over 100 proteins. Just to initiate transcription.

• Promoters: The site where the transcription machinary binds to start transcription.
• The promoter typically facilitates a basal level of transcription
• Typically adjacent to the gene (upstream)
• contains a few 100 nucleotides.
• (31:30) Within those nucleotides there are number of key sequences of the promoter
• TATA Box a.k.a. Core promoter - 25-30 BP region of DNA that is bound by RNA POL. This contians a 7-8 BP consensus sequence. The consensus sequence contains a number of nucleotide sequences and the TATA sequence. This is the RNA POL "docking site".
• mutations in this sequence decrease the level of transcription. Deletion of the sequence results in loss of transcription.
• CAAT Box - these element contain the sequence CAAT or CCAAT located 70 BP upstream of the start of the gene.
  
• Mutations in this sequence=decreased transcription.
  
• (36:15) GC Box - GGGCGG and is found 110 BP upstream of the transcription start site.
  
• both the GC box and the CAAT box can serve as enhancers as well as part of the promoter.
  

• (40:00) Enhancers: Can be found on either side of a gene and can some times be great distances away.
  
• enhancers are typically bound by multiple proteins with a net effect of stimulation of transcription.
• (42:50) How do we differentiate an enhancer from a promoter?
• Promoter regions are found at fixed locations. Enhancers are not found in fixed locations (they can move around.
• You can invert an enhancer without affecting its activity. (not the case for a promoter)
  
• If you move an enhancer to a different gene, that gene will now be regulated by the enhancer.
  
• Promoters are responsible for the basal level of gene expression. Enhancers are necessary for the full expression of a gene.
  
• Enhancers can be cell type specific and promoters are not.

(47:45) How do enhancers stimulate the level of transcription?

• Factors can bind enhancers which help with chromatin remodeling.
  
• When a factor binds the enhancer it bends the DNA bringing the enhancer and promoter closer together. This can help stimulate RNA POL binding to the promoter
  

Genetics Lecture 30, 11/17: Chapter 16, trp Operon, Monod, Yanofsky and Bertrand

lab wed. pre lab write-up is water testing

For Powerpoint slides copy and paste this link in a new window: http://docs.google.com/Presentation?id=dhqwrndc_501cs7scpdq

1953- Monod and coworkers found an operon which is repressible. It was the trp operon.

Trp operon produces enzymes which help the cell synthesize tryptophan.

• (2:35) The presence of tryptophan turns off this operon (trp operon).
• Monod et. al. proposed that the repressor was normally inactive. It was turned on in the presence of tryptophan. This further led them to propose that the repressor had a co-repressor.
  
• What they figured out: the regulation was done with the assistance of constituitive mutants.
  
• 1st constituitive mutant was trpR- strain which produces the trpR protein that functions as a repressor protein
  
• (7:25) 2nd was a mutation in the operator region (analogous to lac O).
• In the absence of tryptophan the trpR repressor protein cannot bind the operator. Because the operator is unbound transcription takes place.
• (9:40) In the presence of tryptophan . . . the trpR repressor protein is made. Tryptophan binds to the trpR repressor. When this happens the repressor binds to the operator and prevents transcription.
  
• (12:20) 5'-UTR - an untranslated region. A sequence of DNA which is transcribed upstream of a gene but not copied. Typically they are involved in gene regulation.
  
• The untranslated region in the trp operon is 162 BP and it is transcribed but not translated. This untranslated region is studied by Yanofsky and Bertrand.

(17:15) Yanofsky and Bertrand (Slide Title: The role of the hairpin in the regulation of the trp operon).

• Irregardless of the presence or absence of tryptophan transcription of the trp operon begins.
• In the presence of tryptophan transcription ceases 140 BP into the 5' untranslated region. (140 of 162 are translated)
• In the absence of tryptophan transcription begins and continues to the end of the operon.
• There is a site between 115 and 140 nucleotides into the 5' untranslated region that serves as an attenuator sequence. (21:50) What happens is that this region can form hairpins in the 5'-UTR RNA.
  
• (23:30) In the presence of tryptophan the attenuator region forms two hairpins. This causes transcription to stop. In the absence of tryptophan the attenuator forms a single hairpin (called the anti terminator), transcription is able to complete.
•