Genetics test 2
Genetics test 2 Bio 330, Genetics
U of L
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This 10 page Class Notes was uploaded by Catherine Montz on Saturday February 27, 2016. The Class Notes belongs to Bio 330, Genetics at University of Louisville taught by Dr. Perlin in Spring 2016. Since its upload, it has received 33 views. For similar materials see Genetics and Molecular Biology in Biology at University of Louisville.
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Date Created: 02/27/16
BIO 330- Genetics and Molecular Biology Professor Perlin Test 2 Set 1 2/11/16 The operon Know before we begin: o The inducer (turns operon on) binds to and regulates the regulatory protein, and the regulatory protein binds to the operator in order for the operon to express o The binding of the regulatory protein to the operator will lead to negative control (ill discuss control below) o What the regulatory protein does, determines type of control o Inducer regulates regulatory protein (no gene codes for this) Negative control Good promoter Transcription is always on, unless negative regulatory protein binds to operator sequence to block RNA polymerase at the promoter Positive control Bad promoter Transcription is always off, unless positive regulatory protein binds to operator sequence, which helps RNA pol.at promoter Operon- a series of genes whose expression is co-regulated by regulating synthesis of their common mRNA. So synthesis regulation, helps regulate the gene expression basically Know that all of these genes are transcribed together Here is a break down of this: You have a piece of DNA. On this DNA (in order) is: RNA polymerase Regulatory protein Promoter Operator Structural genes (a, b, c) RNAP starts running along the DNA After this RNAP, there is a regulatory protein. An inducer could bind to this regulatory protein (remember an inducer is just a chemical reflecting what’s going on in the environment). If it does it alters its shape. RNAP eventually will bind to the promoter and begin transcription The regulatory protein can bind to the operator if there is no inducer located directly after the promoter, however transcription will not occur if it does. If the inducer changes the shape if it binds, then the regulatory protein will not bind, allowing transcription The genes will then be transcribed expressed with the co-regulation of common mRNA Remember the type of control is determined by the type of regulator protein binding to the operator. There are two types of operons: Synthetic - biosynthesis Degradative - breaks a substance down Inducers They make regulatory proteins responsive, they bind to them They can either activate or deactivate them (they are specific) they do this by changing physical conformation of the regulatory protein They do not determine type of control The LAC operon: Metabolism of beta galactoside sugars, for example lactose It is a degradative operon Structural genes involved (z, y, a) and each codes for a particular enzyme o Z-gene: Codes for enzyme, beta-galactosidase Converts lactose into glucose or galactose o Y-gene Codes for enzyme, permease Transport of lactose and other b-galactoside sugars into the cell Codes for protein, which this protein will carry out the rest of the tasks, such as the transport o A - gene Codes for transacetylase Detox of break down products, only if sugar is taken in that causes an issue Another Gene: |Pi | I |p |o| Negative control Degradative Lac I regulatory proteins involved. There will be four of these little proteins surrounding the operator. When lactose is present The inducer, allolactose can bind to the regulatory protein Cause it to change shape No binding occurs to the operator RNA pol. can bind to the promoter Lots of Lactose = this keeps going, transcription happens No lactose = no inducer = regulatory protein binds = transcription will be blocked LAC I regulatory protein: There is four molecules of lac I and they bind to the operator before RNAP can bind to the promoter Can find specific sequence coming from either direction after it jumps on chromosome - Called dyad symmetry After one Lac I binds, it recruits the others to make it easier to find Here is a picture that may help out: So we have a few hypotheses about this binding stuff 1- There are two separate binding sites; shape gets changed 2- There is one binding site; there is competition for the binding site, but if the inducer comes into play, operator can’t bind. EXPERIMENT: There were two classes in the experiment: Mutant I Mutant II And inducer was bound to mutant I, not mutant II. There was no operator binding in mutant I, but was in mutant II. This tells us that there are two distinct sites to bind and the ability to bind inducer is different than that of the operator Problems: To get the inducer in, you need permease. However to get permease you need an inducer! Regulation is not on or off it is on at what is considered high level and on at low levels 1) Since you need permease to bring in the inducer there must be some sort of basal level transcription going on without an inducer 2) Lactose is NOT the inducer, it is actually allolactose High [lactose] - beta galactosidase - glucose + galactose Low [lactose] - “ “ - allolactose Beta- galactosidase is needed to make allolactose and B. Gal is made by the Z gene. 2/16/16 In today’s class we discussed the wildtypes and mutants for the LAC operon and their levels of transcription. We discussed the concept of partial diploids and what is classified as a partial diploid. Let’s get started. Here are some important definitions to know first X-Gal : o Substrate for b- galactosidase o Not an inducer o Changes color to blue/green, which means a high level of expression is on o Stays white/clear when there are low levels of expression on IPTG: o A “gratuitous” inducer o Not metabolized, but is an inducer o It is always induced and it pulls the regulatory protein off of the operator TRANSCRIPTION LEVELS Wildtype LAC + X-Gal (white/clear) Low level transcription with no inducer, only x-gal present Lac I binds to the operator, RNAP is then blocked from binding to the promoter and beginning transcription. However; there are basal levels of transcription still present IPTG (blue/green) With the IPTG inducer present, there is high levels of transcription because there is nothing to block RNAP from doing its job of binding to the promoter and beginning transcription - Mutant (regulatory protein) LAC I : Transcription is always said to be “on” in these guys and there is always a high level of transcription going on with or without an inducer. So with X-gal there is high transcription because RNAP isn’t blocked by the regulatory protein and the same goes for IPTG, its high, but because RNAP can do its job. C - LAC O (operon) - First off, this guy is similar to LAC I . Their phenotypes are the same so you can’t tell them apart from just analyzing the phenotype. But onto the next part, this guy has a little c, which means constitutive. Essentially transcription is always “on”. So when x-gal is present it has high transcription because the regulatory protein isn’t binding to the operator to block RNAP, and for IPTG its high cause RNAP starts transcription Mutant LAC I - LAC I binds to the operator, but it cannot bind to the inducer, so there is low transcription with x-gal and IPTG. With x-gal the LAC I binds to the operator site and doesn’t let go so the RNAP is blocked. With IPTG, it (the inducer) isn’t recognized and it stays bound where it is, and expression cannot perform at high levels. - LAC Z - Less b - galactosidase is made. X-gal appears to be white and so does IPTG so generally by observation we would say they produce low levels of transcription, however this isn’t necessarily true at all times. PARTIAL DIPLOIDS - plasmid introduces part of genome I’m going to make a table for this part so it will be easier to understand Chromosome Plasmid No Inducer Inducer LAC I -- LAC I + Low (white) High (blue/green) No LAC I protein Lac I protein Because protein Regulatory is being made protein is pulled off LAC O C LAC I + High level High level Can’t block Doesn’t/can’t fix expression expression operator because operator mutant it doesn’t recognize operator site LAC O c LAC O + High level Mutant Functional expression Know that: o Bringing in a functional operator on a plasmid has no affect on the operator of a chromosome o LAC I gene codes for protein + o LAC O is only a site that has to be recognized 2/18/16 Remember those little promoter sequences? They’re back! we will discuss them here shortly First let’s trail back to the LAC operon: Know that: o Bacteria will eat glucose first, because it is easier to break down o LAC operon is considered on when it eats lactose o When it is off it eats maltose and galactose Catabolite Repression: This is positive control of transcription, since regulatory protein affects an increase in rate of transcription Catabolite activator protein: CAMP and CAP bind at cap binding site and these will help RNAP transcribe. o Adenylate cyclase makes CAMP, however is glucose is not present, CAMP cannot be made, it blocks the making of it o Glucose is a preferred energy source o Even if inducer is present, if cap binding doesn’t happen, no transcription will happen Glucose is the winner. It’s the best. Trytophan Operon: Biosynthesis of tryptophan (amino acid) it is a synthetic operon and it has negative control. Know that TrpP regulatory protein + Trp is the inducer Transcription in this operon can be brought down to one unit so lets show a break down 600 units 8-10 units 1 unit These units code for 2 trp’s that are adjacent The 14 amino acid peptide is important for this and know the RNA strand is complementary I’m going to give you a break down of how this operon works First know we have the connections that could occur between 1:2, 2:3, 3:4. Ribosomes make trp. If there is something that occurs and these ribosomes are unable to make trp, then an anti-termination signal will be reached and transcription stops at what we call the 2:3. A reason for this stopping may be because there is plenty of tryptophan and there is no need to make more. Another ribosome will come along however and another attempt at transcription will begin right after the 2:3 site. If the ribosome successfully makes trp, maybe there is a starving for trp, then there will be a stem and loop structure that occurs between the connections of 3:4. When there is plenty of trp that has been made then a rho-independent termination signal will become present and stop transcription. Back to the promoter sequences up at the top, In class Perlin specified a characteristic about them you will need to know for the next set of notes. TATA box - known for specificity and its orientation matters CAAT box - known for efficiency and its orientation does not matter GC box- known for efficiency and its orientation does not matter
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