BIO 362 EXAM 3 Review Sheet 2014 Completed PDF

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Download BIO 362 EXAM 3 Review Sheet 2014 Completed PDF

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EXAM 3 Review Sheet: Lecture 21: Transcription Regulation in Bacteria. RNA Polymerase (bound in red this sigma factor sigma 70 vegative sigma factor). Has four Domains, domain 2 and domain 4. Sigma 2 subdomian interacts with the -10 region, sigma 4 subdomains interacts with the -35 region of the promoter. Sigma 4 Subdomain also interacts with another part of RNA Polymerase called the BETA FLAP!!!!! Interactions of sigma 4 with the BETA FLAP, Beta flap is part of RNA Polymerase. When RNAP BINDS PROMOTER sigma 4 interacts with the -35 region , that interaction is what is holding RNA Polymerase at the promoter, you need to break these interactions for RNAP to escape promoter and into Elongation mode. As RNA is made and gets longer then 8-12 nucleotides, it interacts with the BETA FLAP; those interactions disrupt interactions with the sigma 4. So you have double stranded DNA coming into RNA polymerase it is opened up there is a coding strand and a template strand. Secondary channel where nucleotides come in, newly synthesized RNA you also have Beta” Lid, which is thought to separate RNA: DNA hybrid, within RNA exit channel, the RNA then makes contact with the Beta Flap, and these contacts disrupt contact of RNA polymerase with the sigma 4 subunit and this is what is thought to release RNA polymerase from the promoter!!!!!!, it is no longer anchored between interactions of sigma 4 in the -35 region. RNA Polymerase is free to go to Elongation! RNA chain is somewhere in the range of 10-12 nucleotides, during the initial stages of promoter escape, destabilizing interactions between sigma 4 and beta flap and then release of sigma 4 from the BETA FLAP allows promoter escape.

Release of sigma 4 from the beta flap would in turn destabilize interaction between sigma 4 and the -35 element allowing RNAP to let go of the promoter and translocate downstream as it elongates the RNA. Gene regulation. Many different ways to regulate transcription, you can use alternative sigma factors, Positive CONTROL you need an Activator, activator is a protein factor that interacts with RNA Polymerase on adjacent DNA and Activators stimulate Transcription, Negative Control you shut down transcription, protein factor binds to RNA Polymerase or DNA adjacent to promoter and Represses Transcription, you need a Repressor protein to have a negative effect. , You need an activator to have a positive effect, different sigma factors under different conditions bind the RNA Polymerase and enabling transcribe a different set of genes required for different conditions the cell is in. Positive control (An activator that binds Activator Binding Site or an ENHANCER and then interacts with RNA Polymerase, which binds to a promoter region and it enhance Transcription of the Gene, or Genes Downstream. Ex. Cap which is an Activator. A Repressor binds to sequences known as operators, these overlap promoter sequences -10 and -35. OPERATORS ARE REPRESSOR BINDING SITES!!!!!! Those overlap promoters!!!!! . This Binding prevents RNA Polymerase from recognizing promoter, therefore shuts down transcription. Activator UPSTREAM IN THE PICTURE.

In Bacteria there are different Metabolic or Regulatory pathways, you might need multiples genes to get a particular product that the cell needs. Usually they are organized and regulated in Units called Operons!!!! An operon is a set of genes that participate in the same pathway that are regulated by a single promoter, perhaps single activator site, single repressor that is, a set of regulatory elements that control expression of several genes involved in same

pathway. The RNA Polymerase transcribes these genes they are transcribed as polycistronic: several unit genes that are part of the same transcript). Slide shows -35 Region and -10 regions, Start Site. RNA Polymerase recognizes promoter sequences. Proteins that interact with RNA polymerase and regulate activity are called Transcription factors, in Transcription initiation you can have alternative sigma factors, Repressors, activators, So a repressor binds an operator sequence which is usually located downstream of the promoter, overlaps promoter and start of a gene. These Repressors can bind depending on what signals are available, you can have a Repressor that binds DNA operator sequence without a factor (metabolite of some sort) it can bind without Metabolite, but when metabolite binds, it does not interact with DNA!!! You can also have OPPOSITE SCENARIO; you can have a different regulator that requires a small molecule or Metabolite for binding to DNA. (SO YOU CAN HAVE BOTH SCENARIOS) !!!!!!! Ex. Repressor bound to an Operator, but when Repressor bound to small molecule or metabolite it doesn’t bind anymore.

Activators: Usually Bind Upstream, same scenario, you can have a activator that binds DNA, this Binding again can be dependent on some small molecule or metabolite. Why do you need to regulate Transcription? Bacteria grow in an environment that is rapidly changing, they need to respond to these changes in environment, survival depends on their ability to adapt to these environments, change expression of a set of genes so that they don’t run out of food, they can change set of genes to metabolize a different source of food. To do that they express different enzymes that enable them to survive, enzyme synthesis is costly, you have to make RNA,protein, in the process

use a lot of nucleotides and Amino Acids and synthesis is costly. It is ideal not to make all genes at all times, activate genes, proteins RNA when you need them. Can classify genes as Constitutive genes: Genes that are on all the time, usually called House Keeping genes, need these genes all the time. Inducible Genes: Only activated in Certain Conditions. Repressible Genes: Are normally ON but turned off by the presence of a repressor substance. Constitutive: Always expressed or housekeeping glucose- metabolizing enzymes. In Bacteria, Glucose is the preferred choice of energy. Inducible Genes: Made only when needed. Lactose Metabolizing Enzymes are inducible and made only when needed. Transport Enzymes not made for Lactose when Glucose is around, When Glucose is around you shut these genes off. Lac Operon: (A set of Regulatory elements that there is a promoter, operator, Biosynthetic genes that are three genes that are transcribed as a Polycistronic mRNA and their expression is controlled by a Repressor, the Repressor is made from the LacI gene. Normally cells utilized glucose, when glucose run out, cells can transport Lactose, the Lactose is transported using a perm ease , that allows Transport from out side the cell to inside the cell. So Lactose is transported in and then there is an enzyme called Beta Galactodiase, which Hydrolyzes Lactose to Glucose and Galactose, a small percentage of the Lactose is converted to Allolactose (which has a 1’6” linkage. AlloLactose is called an Inducer, meaning it induces gene expression, when allolactose is produced in the cell the lac Operon is activated!!!! People have made Analogs of Allolactose, which can report on activity of Lactose Operon. XGAL when cleaved by B-Galactosidase it generates a color, or signal then you know there is B-Galactosidase activity in the cell. IPTG: Is a Synthetic inducer functions same way as Allolactose. THE LAC OPERON Inducer is ALLOLACTOSE AND IF YOU WANT TO SYNTHEICALLY INDUCE LAC OPERON YOU CAN USE IPTG!!!

Conversion of Lactose to Allolactose is what induces the Lac Operon. KNOW STRUCTURE OF GLUCOSE AND GALACTOSE!!!!!! In a wild type cell, the Lac I gene is transcribed, the mRNA is translated to make LacI Repressor Protein!!!, Repressor protein binds to Operator and shuts transcription off. Lac Operon is regulated by the LacI repressor, which is produced by the LacI gene, this Repressor protein is an allosteric protein meaning it has two binding sites. That is the activity of one binding site is regulated by the activity of the other binding site, it has a DNA binding part, which binds to the Operator. Operator: DNA segment that the Repressor binds to, and then there is an Allolactose Binding site, binding of inducer alters ability of Repressor binding to DNA!!! When inducer-binding site of the repressor is empty then repressor binds to the Operator and transcription is inhibited, when inducer occupies inducer binding, it cannot bind the operator and Transcription takes place. So if no inducer (allolactose) Lactose is not converted to allolactose, the repressor is made and binds to the operator and shuts off Transcription, RNA Polymerase cannot bind. When Lactose is Brought into cell and converted to Allolactose, it binds to repressor and then the repressor cannot bind to operator and transcription takes place. Simple ON AND OFF SWITCH!!! Cell doesn’t have glucose, it then utilizes Lactose! Lactose is brought in, Lactose has to be metabolized, in order for lactose to be metabolize, and you need the enzymes to convert Lactose to the Base sugars. Galactose and Glucose! So how does the Repressor bind and recognize Operator sequence there is an operator sequence that is adjacent to the promoter, there are also additional operator sequences 01 immediately adjacent to promoter, O2 and O3 elsewhere, they have very similar sequences (they are inverted repeats). When Lac Repressor binds it, it binds it as a Tetramer!!!!!!, two Homodimers come together, the tetramer bound to operator sequences binds two operator sequences and creates this road block and occludes promoter, so RNA Polymerase cannot bind and that represses Transcription of the genes

Model on how Tetramer binds, it shows each monomer in a different color, each monomer has a DNA binding part and a dimerization and tetramerization domain. So one dimer binds O1, other binds 02 or 03 and then they interact to form the central roadblock. If you look at the Lac Promoter -35 and -10 Consensus Sequences promoter, it is a weak promoter because it is not close to the consensus sequences. On its own without a repressor or activator, the Lac promoter is a weak promoter. You can play around with the promoter and improve the Lac promoter without any Repressors or Activators. You can articailally improve affinity of RNA Polymerase for this promoter artificially. In the Lac Operon, operator sequences repressor binding sequences overlaps promoter in the 5’ end of the first structural genes.

Operator or Repressor Binding Site overlaps with Promoter and 5’ end of the first BIOSYNTHETIC GENES. Good road block so RNA Polymerase doesn’t have access to promoter and shut off start site as well. Basic Structure of a monomer of a Repressor it has a Helix-turn-Helix Motif, which is important for DNA binding. That DNA binding motif or domain is linked to Inducer Binding Domains and then has a C-terminal Oligomerization Domain, Helix-turn-Helix motif binds DNA, Inducer binding domains bind to the Inducer and C-terminal Oligomereic Structure and DNA Binding activity is affected by what binds at Inducer Binding Domains. Half sites, the Operator, each monomer of the dimer binds to the Half SITES OF THE LAC Operator. O1 operator sequence, one dimer binds O1 and another dimer binds 03 and then it would tertramerize to give you that roadblock. A Helix-turn-Helix part interacting with DNA, another HTH part monomers interacting at different sites. Helix-turn-Helix part makes nucleotide specific contacts with each Half Sites. Each half site interacts

with one monomer of the repressor, DNA binding of one monomer, other monomer binding to each half site.

One dimer interacting with 03 and other dimer interacting with O1, and then they interact with each other. Dimer binding to Half Site of the Operator, other dimer interacts with other half site of the operator, Two operator sequences and then the tetramer binds all four half sites, this binding prevents RNA Polymerase this binding prevents RNA Polymerase from Binding to promoter and shuts off transcription. Now cAMP binds to CAP(CRP) : Cap (CRP) are activator proteins that bind cAMP. When you have allolactose , allolactose binding to repressor LacI , alters its conformation so it can no longer bind to the operator sequence that allows RNA polymerase to bind to the promoter and transcribe downstream genes. If both lactose and glucose are present, Genes required for metabolism of glucose ARE ALWAYS ON!!!!! So if glucose is present the Lac Operon is repressed even when you have Lactose. If glucose and lactose, cell will use all glucose first before going to metabolize lactose. However when glucose runs low, the cell starts making higher levels of cAMP. It actively makes cAMP and lactose is present, lactose is brought into the cell and converted to allolactose and that binds to lacI repressor and prevents it from binding to the operator sequence, RNA Polymerase can then bind promoter, remember the Lac Promoter by itself is a weak promoter, cAMP binds to CAP(crp) and this binding helps RNA Polymerase recognize this weak promoter. When you have cAMP and it binds CRP(CAP) it favors more transcription from a weak promoter. You can have a weak promoter but enhance Transcription by binding of another protein Upstream!!!!! So Activator, it binds to DNA in response to cAMP and it enhances transcription. Normally Lac I gene is Transcribed (TRANSLATED) .Lac I repressor protein binds to Operator sequence and shuts off transcription. If you have Lactose in the Medium and glucose is low!!! (Lactose is converted to allolactose , it binds to Repressor, the repressor comes off, Repressor coming off DNA, allows RNA Polymerase

to make B-Galctosidase which is important for hydrolyzing Lactose , makes Permease which allows Lactose to be brought into the cell, and make Transacetlyase which we don’t know what it does. This is a Polycistronic mRNA each gene has its own Start and Stop Codons, the next gene has it’s own start and stop signal for translation of a particular protein. Normally, in a wild-type cell, the Lac Repressor is made, it binds to Operator, binding to operator prevent RNA Polymerase from Transcribing so you have no Transcription!!!!, Lac Operon is repressed .

When Glucose is Low and Lactose is present, Lactose can bind to the repressor , repressor conformation is altered such that it cannot bind Operator. RNA Polymerase then can go and transcribe structural Genes, then translated to give you proteins responsible for metabolizing lactose. Now you have a Lac I mutant (I-) phenotypically, so the mutant is made , Mutant repressor doesn’t interact with the operator well , so it does not repress!!!!!!! IT DOES NOT SHUT OFF B-Galactosidase ,Permease,Transacetlyase , put Lac I- on chromosome plasmid. Wild type Lac I copy makes functional repressor, in Lac I – when add allolactose it makes Repressor inducible again In Lac I- background , it will not respond to Lactose, it will always be on, you can put wild-type gene and that will restore regulation.

In a wild type setting, normally Lac Operon is off; when glucose is low and lactose is present it is turned on because you make allolactose and repressor comes off. In a Lac I- strain, the operon is on because the Repressor is defective and IT DOESN’T MATTER IF YOU HAVE LACOTSE OR NOT. It is constituvely active, you took an inducible gene and made it constitutive by making a mutation in the repressor, if you complement it with a Wild-Type Repressor it restore functionality and you then have a wild type strain. You can also make changes in the operator, such that he wild type repressor is made but it doesn’t bind. Mutant Oc is an operator mutant; this is again constitutive because repressor cannot shut it down. RNA Polymerase can bind and transcribe that gene, so if you have lactose or no lactose it is the same story, have a faulty operator. when you add allolactose it binds repressor but the LAC OPERON IS ON already. You can have lactose in the medium or you can add lactose. If when genes are off, they are still low level transcripts, you have a few copies of RNA, few copies of permease, B-Galactosidase and bring lactose to cell convert it to Allolactose, If Glucose is present, you don’t make more of these transcripts, when glucose is low, then you need other source of food in this case lactose. Lactose comes in and is converted to Allolactose binds to repressor and induces operon to full scale and make more Permease,BGalctosidase, to bring in a lot more lactose into the medium and induce a new set of genes to deal with the environment of lack of glucose. Lac I is made constituvely You don’t really need a lot of copies of repressor, if there is only one promoter and operator; you only need a few copies of tetramer that will shut operon off. Don’t need a lot of copies to turn on and off a gene.

Have another situation where Is Repressor binds DNA very tightly, your inducer Allolactose , no matter what the repressor does , it does not respond

to Allolactose(ESSENTIALLY CONSTIVELY SHUT DOWN) . Different scenarios where you can have mutation in different parts, either repressor or operator mutations determine whether a set of genes are turned on, induced,repressed, constitive on or off.

Activator Cap or Crp , binds cAMP and enhances ability of RNA Polymerase to transcribe this operon . The Lac I gene mRNA is translated to make Lac I repressor, repressor binds to operator and shuts down transcription of operon, this is absence of inducer. If you have glucose present, inducer will not bind to the repressor. If glucose is low and you have lactose or IPTG present, then you can turn on the operon, Lac I is made, inducer binds Lac I repressor and repressor cannot bind operator, binding now does not interfere with RNA Polymerase, and you can make structural genes that are required for Lactose Metabolism. The Lac promoter Region has two binding sites: RNA Polymerase Binding Site(Overlaps with operator), CAP Binding Site (Has positive effect on transcription of the Lac Operon. No glucose cAMP is released in the cell. cAMP binds to CAP. CAP in cAMP bound form can bind upstream to cap binding site, the CAP-cAMP complex interacts with RNA Polymerase, it enhances ability of RNA Polymerase to recognize weak promoter (Lac operator) . Have to look at context in what the repressor is doing, inducer present or not, if glucose is low, the cell would have brought in more lactose to generate allolactose, the repressor will be off and enhance rate of transcription. Glucose absent

cAMP levels double, camp binds CAP which binds to the CAP Binding site and increase binding of RNA Polymerase for Transcription. It is the CTD of the alpha subunit of RNA Polymerase that interacts with the CAP-cAMP complex in the activator binding site. aNTD interacting with the core of that protein and alpha subunit of C-terminal tail interacts with this activator. CAP bound to cAMP and bound to DNA. When Glucose is present, cAMP levels go down then CAP does not bind to activator binding site and that decreases the affinity for RNA Polymerase and you slow down transcription. When glucose is present, this is one mechanism of decreasing transcription from the Lac Operon. Slowly you will reduce expression of permease, B-Galactosidase and Transacetlyase and reduce influx of lactose into the cell.

Lecture 22: Transcriptional Regulation Transcriptional Regulation of the Lac Operon, archichiture of the lac operon is such that there is a promoter adjacent to it is the first structural gene is the operator sequence and then upstream is the gene encoding the repressor (The LacI gene). When Lactose is being utilized that is Glucose is low. Lactose is brought in, it is converted by this enzyme Beta Galctosidase to Galactose and Glucose and sometimes occasionally to allolactose which is an inducer, when allolactose binds repressor, the repressor no longer binds to the operator sequence, thus permitting transcription of the Lac Operon,

Synthetic inducers (analogs) XGAL is used to look at activity of BGalctosidase or Gene expression in General, if you put in some other gene of interest under Lac promoter or you are looking at Beta Galactosidase activity you can use this and if you want to induce Lac promoter artificially without ...