See the Power Point for Chapter 16 here
http://docs.google.com/Presentation?id=dhqwrndc_501cs7scpdq
Quick Review:
Last time we talked abou the lac operon and how we regulate the expression of the lac operon in the presence and absence of lactose.
Function of the lac operon is to help convert lactose to glucose and galactose.
What happens when both lactose and glucose (primary metabolite) are present?
- ANS: In the presence of glucose the lac operon is off. (fail to transcribe the lac operon because you don't need to break the lactose down)
- so then . . . "How is this accomplished?". It is accomplished through a process called CAP (Catabolite activator protein) and cAMP (cyclic AMP)
- Quick Review: RNA POL on its own has a hard time finding promoters. (That's why we have the sigma factor, it aids the RNA POL in getting to the promoter)
- (3:40) The CAP is always present in the cell. CAP binding to the promoter of the lac operon is necessary for RNA POL to bind a promoter. So without the binding of CAP RNA POL seldom binds to the promoter.
- CAP can only bind to the promoter for the lac operon when its complexed with cAMP. When cAMP is not present, CAP does not bind to promoter.
(7:00) cAMP levels are dependent on the level of glucose in the media.
- cAMP is a derivative of ATP
- One of the enzymes utilized in cAMP production is called adenylcyclase. What happens is that the production of adenycyclase is inhibited by glucose. Thus, when glucose is present in the media the adenylcyclase levels fall. As they fall cAMP levels fall. SUMMARY: in the presence of glucose you have low levels of cAMP, therefore, you rarely form the cAMP - CAP complex, therfore, you rarely transcribe the lac operon.
- Summary of this example: Lactose is present in this example which means lactose is able to bind to lac I bind to the repressor protein, inhibit the repressor proteins ability to bind to the operator. If RNA POL can bind, it can copy BUT the presence of glucose reslts in low cAMP and the lac operon is off.
(11:35 - 21:25) Schau used the following volunteers to demonstrate regulation of lac operon.
Dan - promoter
Becky - operater
Laura - structural genes
Emily - RNA polymerase
Chris - catabolyte activator protein (his hat is cAMP)
Lindsey - Lac I
Lisa - Lac I gene
Britney - Lactose
E.J. - Glucose
(15:25) Review of Monday's lecture:
- Begin with absence of lactose - no sugar at all.
- Lac I gene gives rise to Lac I protein, Lac I protein goes to operator (Lac O). RNA POL goes to promoter and attempts to copy but Lac I stops. As a result there is no transcription that happens
(16:50) Introduce a sugar - Lactose
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator (Lac O). Lactose binds to Lac I and prevents it from getting to operator. RNA POL can then come in, bind to promoter and transcribe.
(18:00) Presence of lactose and CAP - cAMP complex.
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator
(Lac O). Lactose binds to Lac I and prevents it from getting to
operator. CAP helps RNA POL find the promoter.
(19:10) Presence of lactose and glucose and CAP - cAMP complex.
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator
(Lac O). Lactose binds to Lac I and prevents it from getting to
operator. CAP helps RNA POL find the promoter BUT glucose reduces levels of cAMP and without it you can not get RNA POL to the promoter.
side note: Lac I repressor protein (when present and functioning) binds to operator and inhibits RNA POL from transcribing genes
(23:15) 3 different processes we NEED to KNOW
- absence of sugar
- presence of lactose
- presence of lactose and glucose.
- For each of the above conditions we should know
- Is the operon transcribed?
- How does the regulation of the operon take place?
- In regards to regulation include talking about cAMP and CAP and lac I repressor protein
(25:40) Additional work by Jacob and Monod
- Quick Review: The understanding of how this regulation takes place relied on these two constituitive mutants: lac I- and lac O-
- When these above mutations were orginally identified, nobody knew if they were mutations in cis-acting elements or trans-acting factors. We (as a genetics class) know that Lac I is a trans-acting factor and Lac O is a cis-acting element. Jacob and Monod did not know this as a result they developed the "Cis-trans test".
- (28:10) "Cis-trans test", used to determine the nature of these mutations. How they did the test:
- The lac I mutant strain in the cell constiuitively expresses the lac operon.(despite presence or absence of lactose. Normally it is sensitive to lactose, however)
- Sooo . . . They placed a wildtype (WT) copy of the lac I gene onto a plasmid (a piece of extra chromosomal DNA). Then they introduced this plasmid into the lac I- strain. What this accomplishes: The WT copy of the lac I gene on the plasmid will produce or give rise to the WT lac I protein. It will NOT correct a mutatnt lac I DNA sequence in the cells DNA.
- Important: The presence of the plasmid with the lac I gene can correct a mutation in a trans-acting factor. Not in a cis-acting element.
- (33:35) What will the presence of WT lac I protein do to the lac I mutant strain?
- We would expect that this would correct the constituitive phenotype. So the lac I mutant strain with the WT copy of lac I on a plasmid, making protein, will no longer be constituitive.
- Now that we have a normal protein it will require lactose to bind to it to prevent it from binding to the operater. After performing the test the conclusion is that lac I produces a trans-acting factor - a molecule that is a protein that is produced to regulate the expression of another operon.
- Summary: This cell will only express the lac operon when lactose is present
(36:55) Now they want to know about lac O.
- The lac O mutant strain in the cell constiuitively expresses the lac operon
- Is lac O a cis - acting element or a trans acting factor?
- Introduce a WT copy of the lac O sequence on a plasmid, this can produce WT copies of the lac O protein. However, the problem is, lac O does not produce a protein.
- The presence of the plasmid does not correct the mutant lac O sequence. Therefore, the lac I repressor cannot bind to the mutant lac O sequence.
- The presence of the plasmid does not correct the constituitive phenotype.
- Thus we conclude that lac O is a cis-acting element.
- (plasmid will correct a trans-acting factor but not a cis-acting element.)
(43:25) Recap of Jacob and Manod "Cis-trans test"
- cis acting element - sequence of DNA that is acted upon by a trans-acting factor to regulate the expression of a gene or operon.
- trans acting factor - protein that regulates the expression of a gene or an operon by acting ON a cis-acting element. Binds to DNA and either turns on or off the expression of a gene.
- In the lac operon, the binding of lac I (the repressor protein) to the operator turns off transcription. When you mutate either of these elements you mutate the protein and it won't bind to the operator if you mutate the operator the normal protein can't bind - same effect. The end result is constituitive expression - we always express the lac operon.
- (46:50) What Jacob and Monod started this test, all they knew was that they express constituitively. They wanted to know was it trans-acting factor or cis-acting element.
- They take the mutant strains, started with lac I. They have an abnormal copy of the lac I gene.
- They go to a different cell and get a normal copy. They put this normal copy into the lac I mutant strain. It will be transcribed and you will make WT copies of the lac I protein. So instead of a cell that only has "bad" versions of lac I protein that can't bind to the operator, we have a cell that also has normal versions of the protein that can bind to the operator. When that happens the cell that was always expressing the lac operon is now only going to express the lac operon when lactose is present - so there is a change in the phenotype of the cell. BECAUSE that works for lac I we say that lac I is a trans-acting factor. If it DID NOT work we would have called it a cis-acting element.
- (50:00) They then repeat the process for lac O.
- They take a mutant copy of lac O and put in a normal version (from some other cell). It is a cis-acting element.
