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28 Cards in this Set
- Front
- Back
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Diploid analysis was done to identify the functions of the parts of lac operon. F'lac is the name of the plasmid introduced in bacteria. |
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Overview of what we talked about last week |
We have a scenario where we have a repressor that is synthesized. It sits on the operator, turning off lac op. When we have alla present, that molecule is the inducer. When it binds the repressor, it causes a conformation change that no longer allows it to bind DNA so it loses that function. In that case, RNA Poly can assemble and we get transcription of genes in Lac Op. |
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1st scenario |
B. OFF No lactose, therefor no inducer. Anytime glucose is present, the cell will want to metabolize that and keep its lac operon off. The repressor is sitting at the promoter region. |
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2nd scenario |
B. OFF. No sugar and still no inducer therefore no allo and the repressor will bind the promoter, inhibiting transcription of the lack operon. We would just be synthesizing a diff sugar at this point |
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3rd scenario |
B. OFF It wants to be off cuz even though allo is present and this will be removing the rep from the promoter region, we have glucose so we don't actually want to turn on the lac operon. So this becomes a scenario where we get a low basal level of expression. What we want to do is to bring other factors to boost the expression and make a lot of something. |
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4th scenario |
A. ON In this scenario we actually want to turn the lac operon on to it's highest level. We want to ramp up expression of genes that metabolize lactose. |
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Negative regulation of the lac op is a function of the rep and how we only turn the lac op on when we remove the rep. Positive regulation occurs at the same time. We have an activator that interacts with the poly near the promoter to induce higher level of gene expression. |
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CTD tail interacts with upstream elements. Here, the tail interacts with CAP. CAP (CTR in book) can sit on the DNA and interact with tail. Interactions with tail and poly can have strong effects on expression. They can increase/decrease rate of expression. |
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How is cap regulated? |
It's regulated through action of cyclic AMP. The concentration of camp is going to be high when you don't have any glucose. Here we see glucose inhibits enzyme that makes CAMP. There will be an inverse relationship between glucose and camp levels. |
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We have glucose absent in cell. Glucose comes in through this transporter, adenylylcyclase is also on the membrane. When it's absent, we'll have higher levels of camp made, when glucose is present, it inhibits camp. |
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How does CAMP regulate CAP? |
It does so through binding it and changing the ability of CAP to bind DNA. CAP requires the presence of camp to bind DNA. CAMP bound to CAP changes the shape of CAP and allows it to bind to DNA. |
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If we have a lot of glucose present, we're going to inactive adenylyl cyclase (ADC) therefore well have low levels of camp. CAP wont bind DNA cuz there isn't enough camp for it to do so. Here we'll get infrequent transcription of lac op. We can get a high level of expression of genes if we have low glucose. So once we run out of glucose, we re-activate ADC, boost CAMP levels, CAMP binds to CAP, CAP binds DNA and CAP induces high levels of expression of lac operon |
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When we have high glucose and low lactose. Where the rep leaves the operator but we can't get rna poly to associate with cap so we wont get high gene expression. High glucose + lactose, we will have repressor removed and rna poly located at promoter but won't get the added boost of having cap bound to dna to induce that Low glucose + high lactose. Will get high levels of expression. Will catabolyze lactose present in high levels, rep is always off DNA, CAMP will be made, binds CAP, CAP binds DNA and interacts with rna poly and will get positive regulation turned on and negative regulation turned off. |
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Transcription Factors |
Transcription factors are any proteins that binds DNA. They usually have at least 2 domains, 1 allows them to bind DNA and the other to interact with something. Can have DNA binding domain and activation domain. |
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C. Lac Operon will always be turned on, only if lactose was present. Not dependent on glucose anymore. When rep is moved cuz allo is present, RNA poly is fused to cap so tail is touching CAP, so we get induction of lac operon. |
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B. If lactose is absent -- Repressed by lac repressor If lactose is present -- Basal expression only |
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A) |
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C) Lactose is present so we have no negative regulation, which means we'd get a low level of lac operon expression D) Lactose is present which means negative regulation is off. Highest level of expression |
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This is positively regulated by CAP and CAMP. Same scenario where as long as you have glucose, you're going to want to have this turned off. It's only when you don't have glucose & do have arabinose where you do want the arabinose catabolic enzymes expressed. |
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The regulatory molecule is going to bind different places of DNA, rather than binding dna and coming off of it. The regulatory molecule AraC is always going to be binding DNA, just depends on where in the dna it binds. |
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When AraC is not bound to arabinose, meaning arabinose isn't present, it binds 2 separate regions causing a bending of DNA and blocking transcription. In the presence of arabinose, it changes it's conformation allowing it to bind a region that will allow it to enhance the expression of arabad operon. |
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D. Negative regulation is removed, positive regulation is present. |
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This is an operon for trp synthesis. When you synthesize trp, that means you've run out of it so these genes are only going to be on when you have a complete lack of trp. We have a similar scenario here where we have a repressor protein. Rep only bind op when its bound to trp. It's going to sit on op and block transcription of genes that lead to enzymes that synthesize trp. trp rep is "leaky" meaning it doesn't work well, so it's supposed to bind DNA but the fit isn't perfect. It only turns off partial expression. The cell has a method to overcome the fact that the TRP rep sucks. It happens by the leader sequence. Their is a region within the sequence called the "attenuator" which means to "shut something off." So under high trp levels, we'll make attenuated mrna which is much shorter than the mrna we want to make when we actually have low levels. Under low levels, we synthesize how mrna. Under high levels, we'll only synthesize the atenuated region, it will stop at atenuated region. *If you're making something, you're only going to want to make it when it's not present. *If you're breaking something down, you're only going to want to turn it on when that thing present |
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Zoomed in to leader sequence |
The mRNA has 4 different regions. Region 1 has 2 adjacent trp codons early on in sequence. If we were a ribosome translating mrna, and there was a depletion of trp in cells, once it reaches these trp codons, the ribosome will stall. This has an affect on how mRNA folds. When mRNA exits Poly, they can rapidly fold into 3D structures. Plenty of TRP makes it so ribosome doesn't stall and it will stop at the STOP codon. Leader peptide is the small region that gets translated. |
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Different ways leader sequence (LS) can fold |
Under low trp, ribosome will pause at trp codons. When this happens, regions 2 and 3 pair up as they exit the polymerase, before region 4 is synthesized. When we have high trp, we stop at the end of leader 1 sequence. By doing so, the ribosome blocks part of region 2, meaning regions 3 and 4 will pair. There are a string of Us at the end region 4, so when regions 3 and 4 base pair with the string of Us, you are actually going to get transcription stopped at that point. This affects rna poly. When regions 2 and 3 pair, there is no string of Us so it doesn't function as an attenuator for rna poly. This scenario doesn't stop the poly so rna poly will keep going and make full length mRNA. |
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Different view... |
The 1st is where there is no ribosome present. 2nd scenario has low trp. There will be 2 adjacent trp codons which will cause a stall because it has to wait for trp tRNA, but we won't have these tRNAs when we have low levels of trp. Then regions 2 and 3 come out of poly, and pair up. Region 4 is then synthesized but we don't have the attenuator sequence that stops transcription. Instead rna poly is still associated with dna, it will move forward and just downstream are the trp genes. 3rd scenario: We have many trp, the ribosome can go through them and stop at stop codon, partially blocking region 2 which prevents regions 2 and 3 from pairing. This prevents rna poly from synthesizing any more rna. Only the leader sequence would be transcribed in this scenario. |
