Ch. 12 - Regulation of Gene Expression Ch. 13 - Global Regulation (Exam 2)

TRYPTOPHAN OPERON - types of gene regulation 1) TrpR Repressor what type of repressor is it? what does it do and what is the result? what type of control and regulation is this?

- is an aporepressor (= repressor of negatively regulated biosynthetic operon that is active only if corepressor is present. Aporepressor + corepressor = repressor - TrpR binds as a dimer. - see Box 21.1 re: helix-turn-helix (HTH) motif - negatively regulates more than trp operon (also trp, trpR and aroH [biosynthesis of aromatic amino acids]) Note: TrpR negatively regulates its own expression --> autogenous control / autoregulation

what is Signal Transduction? what does it rely on? on what side does autophosphorylation take place?

- two-component regulatory system - relies on two proteins: sensor, response regulator sensor - sense what is environment and it is a protein that spans the cytoplasmic membrane (like 7 times). When there is a stimulus that the sensor senses, this protein is a kinase and phosphorylates (1st and auto and then it can transfer the protein to a response regulator). Autophosphorylation takes place on the cytoplasmic side

Additional Types of Gene Regulation for Trp other than TrpR Repressor

2) end-product inhibition / feedback inhibition (Trp inhibits first enzyme in Trp production pathway - not affecting transcription just the activity of the enzyme) 3) attenuation

Galactose (gal) operon Q: How to increase chance of picking up galO mutations (since most would occur in galR)?

A: Add an extra copy of galRs (so there are now two copies)!

Q: How do you know lacI- isn't a trans-acting lacZ or lacY?

A: Because it doesn't complement either lacZ or lacY.

Q: How to tell the difference between (+) and (-) control systems?

A: Effect of mutation in regulator protein.

Q*: Why not just increase amount of repressor?

A: If did, would not have the 0.1% basal expression necessary to make LacY, which is necessary to bring lactose into the cell. Think about what happens to lac operon in absence of lactose in presence of small vs large amounts of LacI. If you have 10 lac repressors you get 5 beta gal per cell Increasing the amount of repressors in the absence of the inducer means you will have less beta gal in the cell - if you make less beta gal, or 0, then you cant induce (too much repressor means less beta gal which means less inducing)

Q: Why does it make sense that the lac operon has a weak -35?

A: bc wouldn't need CAP-cAMP activation [lac would be on even + glucose]. A problem of this would be that slopes of two growth phases should be different

GENETICS OF lac OPERON - EXPERIMENTAL RESULTS (A) Complementation tests with lac mutations --> Lac- mutants Q*: Where are dominant Lac- mutations located?

A: in lacI such that a super repressor (because it represses even in the presence of lactose) is created (i.e. it does not bind inducer) --> lacIs Cant block the mutated one Lac z and y make betagalctocidase and it would be possible if they were oligodimers that they could in fact affect transcription (however this isn't the answer) The answer is to get lac-, the mutation would have to generate a non-inducible phenotype. Which means it doesn't act as the WT would and would generate transcription only in the presence of lactose. Which means that I wouldn't be turned on all the time, because turned on all the time would mean the op was mutated (lac+ = op). Therefore, the mutation would have to be in lacI. You would have to mutate LacI so it no longer recognized lactose (it was still in a trans acting gene but the repressor couldn't recognize lactose). This is very rare

What does CAP do? How does CAP work?

A: is a positive control factor A: depends on where it binds; either facilitate formation of closed complex (via interaction with a-CTD, or formation of open complex (via interaction w/a-NTD)

Q*: What kind of mutation with (+) control would be constitutive?

A: those that change activator protein to activate without binding inducer

Alternative Sigma Factors? how many sigma factors does E coli have?

All promoters recognized by various sigma factors in E. coli have same size and location with respect to +1 start site (i.e. -35, -10) except s54 (-24, -12). E. coli has fewer than 10 sigma factors (rpoS= ss; rpoN= s54; fliA= s28/ sF); B. subtilis has >20 (are involved in spore formation) recognize different sets of promoters to control expression of specific groups of genes

Q: what genetic strategy was used to isolate galO mutants?

An extra copy of galR was added to the genome because most galO mutations occur in galR.

How does it make the heat shock proteins in 10-15 minutes and then still have the ability to turn them down without an environmental stimuli Chaperones - help proteins properly fold how many copies of rpoH are there at low temps? what happens at high temps? what is this change due to? what is autoregulated and what is not? where does this regulation occur? what does the heat shock temp affects? what does the increasing of the temperature then do?

At low temp, are few copies of RpoH (s32) At high temp, see 15X increase --> only see small increase in transcription (rpoH has 4 promoters, but none are s32 promoter) Increase due to post-transcriptional control of s32 1) Translation control - increased translation of mRNA 2) Increased protein stability/activity (heat shock and the sigma protein) rpoH is NOT transcriptionally autoregulated s32 likely translationally autoregulates (represses own translation) Has 3 sigma 70 promoters and the 4th is another sigma factor All a promoter does is recruit rna pol Heat shock temp affects h bonding and this can affect the RAN secondary structure (SD hairpin) Increasing temp and get translation of RNA made by sigma 70, however at high temp you only see a small increase in transcription You arnt making more transcript but you are regulating post-translationaly

Attenutation is associated with what operon? what occurs and what characteristics does the process have?

Attenutation - trp operon

Why two promoters for gal?

Because still need gal expression even in presence of glucose - PG1 is catabolite sensitive; PG2 is not (is repressed by cAMP-CAP) Two promoters; two operators; two repressors (either works) Gal is a catabolite sensitive operon, but catabolite repression needs to be different than with lac. GalS primary role is regulating Gal transport system.

Other relevant characteristics re: catabolite sensitive operons Q: Why does this make sense?

CAP-dependent promoters do not have a good -35 sequence, and some lack a good -10 sequence Because they have CAP to stimulate expression.

System to allow expression of multiple genes to be tied to [glucose]

Catabolite repression = system by which cell uses best carbon energy source. says that it will sometimes repress enzymes for one even though the catabolite is present Catabolite sensitive operons = those subjected to catabolite repression KEY to catabolite repression is that [Glu] inversely proportional to [cAMP]

describe class I, II, and III mutations where do they occur? what type of lac mutants are commonly used in the lab?

Class I mutation mutates CAP binding site (= weak Plac; fairly insensitive to [Glu]) Class II mutation in -35 (reduces RNAP binding, still catabolite-sensitive) Class III mutation UV5 makes perfect -10 sequence (strong Plac, no longer need CAP) (so is a Class I suppressor) --> lacUV5 mutants are commonly used in the lab

Constitutive = Noninducible =

Constitutive = always ON Noninducible = always OFF

why did bacteria develop quorum sensing? what is it good for and why? what is this an example of how it is advantageous? what do different species of bacteria generaly used to distinguish self from non-self? what question does this pose?

Developed this to determine what was around them (other bacteria, same bacteria, different, etc) Good for pathogens because they don't start infection until they know it will be successful (when they reach a certain conc) --> example of how it is advantageous to know what is around the bacteria Different species and genre of bacteria commonly use the same things to sense self from non self (1st 3 are used 99% of the time) If using the same signal then how can you distinguish yourself from others? (same species to nonspecies)

How is s32 protein stability/activity changed?

DnaK (a Hsp) binds s32 . When bound: - s32 is more susceptible to protease (helps it degrade) - s32 is less active in transcription (blocks sigma 32 from enhancing transcription) These are at low temp DnaK, also a chaperone (helps proteins fold properly) At low temp, DnaK binds s32 --> DnaK-s32 is more susceptible to protease DnaK- s32 is less active in transcription (DnaK blocks RNAP- s32 formation) At high temp, see increased amounts of denatured proteins. Chaperones act on denatured proteins under heat shock. Because DnaK is binding denatured proteins, is less to bind s32 --> get less s32 degradation and less rpoH transcription inhibition.

Q: How does LacI block transcription?

Doesn't block transcription by blocking RNAP from binding but actually recruits RNAP binding RNAP is a closed complex and is bound to the promoter but hasn't unwound anything It keeps it in a closed complex This means this system allows a faster response to a inducer because the pol is already sitting there (RNAP doesn't have to find a promoter, it already has because the repressor has already recruited it to bind, but just doesn't allow it to take the next step)

what do E. coli prefer as a carbon source?

E. coli prefer glucose as a carbon source.

How LacI works..... what is bound and in what combinations? what does full repression require? why? what is the main binding site?

Each dimer binds an operator. There are two secondary operators (O2 & O3). If remove O2 or O3, get 2-4X repression reduction. If remove both, get ~50X repression reduction. O1 is the main one and is right beside the operator ßLacI binds as a dimer; acts as a tetramer Full repression requires forming tetramer Because when it binds an operator it brings the 2 together The looping out allows the tetramer to work (better than 2 dimers) 1 can bind to 3 or 1 can bind to 2 but not 2 and 3 (not sure if 1,3 enhances pol binding more than 1,2) All this allows pol to bind and actually helps it bind

what are the different transcriptional regulatory systems? what are the effector molecules and what are examples of each? how does each effect gene expression?

Effector molecule category based on response to regulator molecule: a) Inducible = regulator molecule increases gene expression (i.e. need regulator molecule for expr.), operon functions only in presence of small-molecule inducer b) Repressible = regulator molecule decreases gene expr. (i.e. get expr. only in absence of corepressor); operon transcribed only in absence of corepressor. For negative control the gene would still be transcribed but the expression would be turned down. For positive control the gene default is off unless the activator turns it on

what can effector molecules do? what are the different types and what makes them this way?

Effector molecules can increase or decrease the regulators. Effector molecule, with regardless to pos or neg control would increase expression this would be an inducer, if decrease without anything else it would be a corepressor

what is occurring when RNA pol pauses at the 1:2 pause site?\what occurs if Trp is absent? in what organisms does this occur? what is this called?

Pol is transcribing and region 12 hairpin Ribosome is loaded onto the nascent RNA before region 3 is transcribed The ribo stalls where it is occupying region one 2 is unaffected by the ribosome and 23 forms the hairpin before 4 is transcribed and you still get transcription This only happens in bacteria because you have co-transcription and co-translation Only in bacteria will translation be able to affect transcription ATTENUATION

Mechanism of [Glu] and [cAMP] what is a problem of this and how is it answered?

Problem: how to prevent basal lac expression when glucose is present? A: inhibit lactose transport

Example of signal transduction

Some pathogens use two-component system to sense when they are within certain host tissue cells. Basis: MAGENESIUM CONC. à [Mg] is generally lower inside cells than at external sites. à allows activation of genes that facilitate survival against host defenses When Mg conc is below the threshold it turns on other genes and this is a way the pathogen survives on the inside

what is Coordinated control? what is targeted during this?

when you change environment the cells changes the expression of more than one gene Operons are targeted during this because you have one promoter which controls the expression of multiple genes

GENETICS OF lac OPERON - EXPERIMENTAL RESULTS WHAT TEST WAS PERFORMED? how was the experiment done? what did Jacob and Monod find? what was there interpretation?

(A) Complementation tests with lac mutations --> Lac- mutants Experiment = merodiploids of (i) wt and (ii) mutant à growth +/- lactose? Jacob and Monod found most mutations were recessive to wt Interpretation = those mutated genes encoded something required for lactose utilization When those recessive mutations were complemented with other recessive Lac-, they sorted into two complementation groups (lacZ and lacY) (second copy makes what is needed and you don't need the mut copy) Other recessive mutants were rare: - some recessive mutations (on F') could NOT be complemented with a lacZ- or lacY- chromosomal mutation.

GENETICS OF lac OPERON - EXPERIMENTAL RESULTS INTEPRETATION WHAT IS THE EXPERIMENT? Q: If mutant is recessive, what result would be expected? (i.e. would there be growth if lactose is sole C source?) Q: If mutant is dominant, what result would be expected? (i.e. would there be growth if lactose is sole C source?)

(A) Complementation tests with lac mutations --> Lac- mutants Experiment = you make a merodiploids (partial diploid) and gorw them in the ab or pres of lactose. of (i) wt and (ii) mutant à growth +/- lactose? A: get wt phenotype (do get growth on lactose [=Lac+]) - inducible. Then it would be like the wildtype and be inducible A: get mutant phenotype (do not get growth on lactose [=Lac-])

GENETICS OF lac OPERON - EXPERIMENTAL RESULTS (B) Complementation tests with lac mutations --> Lac+ mutants are these mutants inducible or constitutive? Q*: What is the wt and mutant phenotype?

(constitutive mutants) Results: All recessive mutations were in (trans-acting) lacI. Inducible because still have one good copy

Basics of lac operon what does it produce? what does it contain? it is made as what? what does lacl make? what is considered to comprise the lac operon? what inactivates the repressor protein?

- lac operon produces proteins that bring lactose into cell and break it down into glucose and galactose - contains three structural genes (lacZ = b-galactosidase, lacY = permease [allows lactose to enter the cell], lacA = transacetylase - seems to have no important funciton). - made as one polycistronic message - lacI makes repressor protein (is a regulatory gene) - together, lacZ, Y, A and lacI are considered to comprise the lac operon - allolactose inactivates repressor protein

Q: What would happen if mutate DNA so region 2:3 cannot form? (= trpL75) Q: What would you expect if start codon was changed to AUA (trpL29) such that translation doesn't start? Q: The cell is starved for another amino acid. What is the affect on trp operon?

---> see transcription termination even in absence of tryptophan ---> see transcription termination even in absence of tryptophan ---> prevents attenuation (depends on location of other amino acid codon)

How does [Glu] impact [cAMP]? what is given? what occurs if [Glu] is high? what about when it is low? how is pyruvate ratio affected?

1) AMP --> cAMP done by adenylate cyclase (AC) 2) AC is active only when phosphorylated 3) P on AC comes from IIAGlc component of PTS (= PEP [phosphoenolpyruvate]-dependent sugar phosphotransferase system) 4) P on P~IIA comes from Hpr-P (histidine protein) 5) P on Hpr-P comes from PEP 6) PEP:pyruvate ratio is affected by presence of rapidly metabolizing substrate. NOTE: P = phosphate If [Glu] is high, PEP: pyruvate ratio is low. If PEP: pyruvate ratio is low --> no Hpr-P --> no P~IIA --> no AC activation --> no cAMP If PEP: pyruvate ratio is low --> IIAGlc is mostly dephosphorylated (is transferring P to Glu), and IIAGlc inhibits LacY permease If [Glu] is low, PEP: pyruvate ratio is high. If PEP: pyruvate ratio is high --> have Hpr-P --> have P~IIA --> activate AC --> make cAMP --> bind CAP --> increase lac expression If PEP: pyruvate ratio is high --> IIAGlc is mostly phosphorylated (not transferring P to Glu), and P-IIAGlc does NOT inhibit LacY permease (so lactose is transported into cell)

Specificity of Protein-DNA Interactions?

1) genome size (target vs nontarget sites/size) 2) protein specificity 3) amount of protein 4) total number of target sites

co-transcription-translation steps if Trp is present

1) high levels of tRNATrp are present 2) ribosome continues translating until it reaches stop codon on TrpL (Note: ribosome overlaps part of region 2) 3) RNAP continues transcribing RNA regions 3 & 4 while ribosome is stopped at UGA, hence 3:4 forms. *all of this is if trp is present (these particular steps)

co-transcription-translation steps if Trp is absent

1) low levels of tRNATrp 2) ribosome will stall at site before stop codon à region 1 is covered but not region 2 3) when region 3 is transcribed, it will form hairpin with region 2 (before region 4 is made) 4) transcription continues

Specificity of Protein-DNA Interactions Parameters that affect trans-acting factors control of gene expression. how do each work? what are these parameters important for?

1)genome size (target vs nontarget sites/size) 2) protein specificity 3) amount of protein 4) total number of target sites Important for anytime you talk about a protein and a target on DNA

describe what occurs when E. coli has more than one carbon source. what type of growth is this?

2 carbon sources, glucose and non-glucose It uses up the glucose first because it is the preferred carbon, it uses all the glucose and then has another lag, this lag is the cell changing the proteins being made because it no longer needs the cells to metabolize glucose but needs to to metabolize another carbon source (galactose, lactose) This slope for the other carbon source is not as high as the one for glucose Uses best first and then adjusts to the second best and grows slower (but still allows it to grow)

what is a genomic library? what are the steps and what cell grows faster?

= population of host bacterial cells, each of which carries a DNA molecule (plasmid) that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the genome of the source organism. https://www.youtube.com/watch?v=GJ6eVSwZf0g The cell that takes up the tsp mutant is the one that grows faster (introduce the plasmid into a temp sensitive mutant cell) Watch video for the steps

Q: How is repression rapid if it occurs by DNA-bound repressors finding target site?

A: Is not random association-dissociation (would be too slow), must be facilitated (somehow) They arnt just floating around the cell hoping to bind, so there must be some sort of facilitated process (just don't know how)

Q: Makes sense that Trp would regulate trp operon, but not negatively regulate trpR. Wouldn't you want more TrpR in presence of Trp?

A: Maybe this system ensures faster repression of trp operon (as will make more TrpR in absence of Trp, so that when Trp is added, get quick shut off of trp).

Galactose (gal) operon Q: How to isolate an O mutant? (problem: get expression even in absence of Gal) what experiment would be done?

A: Need to take advantage of galRs mutation (super repressor). Experiment: place galRs strain on medium containing ONLY galactose and look for growth

Q*: Does mutation to constitutive expression suggest wt gene is under (-) or (+) control? how large is the operator? which type of mutation is most likely to happen (where the mutation occurs)

A: Negative (any mutation that inactivates the repressor would be constitutive. If the gene is always on, then the mutation would have to repress the gene. Therefore, the WT would be under negative control because the gene is transcribed continuously and negative control means the gene is always turned on (WT is negative and the Mut would be pos) The operator is ~20 bases and the typical gene size in bacteria is about 1000 bases (E.coli genome - 4 million, with approx. 3000 genes). The continuous transcription of a gene, as opposed to facultative expression, in which a gene is only transcribed as needed. A gene that is transcribed continuously is called a constitutive gene. Can activator cant bind the UAS if the corepressor is there or the binding site isn't there The inducer and corepressor have a binding site and mutating this would affect binding as well as mutating the repressor gene Mutation to the activator gene is rare but one to the repressor gene is more likely to happen (target site much bigger)

Q: Why not eliminate repressors/activators and just have inducers act directly on DNA/RNA?

A: That would require that inducers bind DNA or RNAP which would reduce flexibility of range of interactions between inducers and their corresponding transcription factor. More flexibility in being able to bind a poly pep rather than a nucleotide This allows for more flexibility regarding environmental changes and allows the bacteria to make more changes (more control and flexibility for the ability to make changes)

What is cAMP? What does cAMP do?

A: binds to CRP (cyclic AMP receptor protein) a.k.a. CAP (catabolite activator protein) A: is an inducer; activates CAP

Q: How to determine, using genetics, whether operators operate independently or in cooperation? what are the results? what are the conclusions?

A: compare expression in single vs. double mutants Result: double mutant expression is greater than sum of single mutant expression. Conclusion: O's act synergistically

Q: What is the advantage in the gal operon having 2 promoters?

A: galactose is used as an energy source as well as to make polysaccharides, therefore, having more than one promoter allows for increased expression of this gene for energy purposes as well as the generation of products 2 promoters because still need gal expression even in presence of glucose; Two promoters; two operators; two repressors (either works) Steck answers -It allows gal expression even during catabolite repression -Allows finer control of gal expression, which is important because gal products are used for multiple purposes -It allows one to be used while the other is being subjected to catabolite repression -Each can have a different role/control mechanism -If there are 2 promoters they have different control mechanisms and respond differently to different stimuli (more than likely these are not 2 strong promoters)

what do chaperones normally act on? what is DnaK? how is it involved in s32 protein stability/activity? what does s32 help do? what is the main role of DnaK in this process and what does it ultimately allow for? what does all of this together explain?

Chaperones normally act on only completed polypeptides. DnaK is a chaperone and if there are not as many denatured proteins there isn't as much to bind sigma 32, not as much is degraded and it is available to enhance transcription as well Sigma 32 helps transcription and more DnaK negatively regulates its on transcription as more is made during the first minutes of the heat shock TAKING CARE OF THE DENATURED PROTEINS (WHAT DnaK does) - how there is an increase All this explains why there is a 15 or so minute gap where the cell is transitioning to the environment and why there is a change in protein expression

what are the 2 purposes of a complementation test?

Complementation Test Two purposes: 1) To determine if mutations are in same or different gene - as first discussed in Ch. 3 - put two mutations in same cell --> restore wt phenotype? 2) To determine if mutation is dominant or recessive - add mutated gene to wt cell --> do you see the mutant/wt phenotype when they are together in the same cell? - this is what Jacob & Monod did with Lac mutants

Generic attenuation model what occurs? wjat is the connection based on? what forms and how can this occur?

Connection based on if the ribosome stalls on the Hairpin can form in the leader region and if it doesn't the gene is folded and you get transcription termination B - 3 regions that can form 2 diff hairpins If BC hairpin forms it will cause the pol to fall off The AB allows for it to continue

REGULON example? how were components of this regulon ID'ed? what occurs and how is this used? what is the gene that complements?

Example: heat shock/stress response, and s32 If shift E. coli from 32o to 40o, the synthesis of ~30 proteins is abruptly increased and then decreases to pre-shift levels after ~15-20 min. - Makes a bunch of proteins to adjust to environment and then after it has adjusted it stops making as many products --> the proteins made are called heat-shock proteins (Hsp) --> they are made as part of the stress response Components of this regulon were ID'ed via temp-sensitive mutants. --> at restrictive temp, no Hsps were made (just didn't make the 30 proteins) Introduce members of E. coli genomic library into the mutant cell, then ID what complemented the ts mutant (i.e. characterize those that grow at 42o). How they found where these ts mutations were. The gene that complements is htpR (rpoH). Rpo = rna pol and H = a sigma factor affects binding ability of pol and allows it to find a promoter Sequencing reveals this gene encodes a sigma factor! This directs the pol to a subset of genes (the hsp genes)

Examples of how QS is used.

Examples of how QS is used. a) pathogens coordinate attack only when they reach a critical concentration (animal and plant pathogens); QS systems control expression of virulence factors b) to synchronize cellular functions that facilitates mutual existence; for example - QS systems control motility, exopolysaccharides production, biofilm formation, symbiosome formation, plasmid transfer, root nodulation, and N-fixing efficiency in Rhizobium. c) diffusion sensing (determine how likely an exofactor will diffuse away) - Bacteria release enzymes that degrade food, so if you are going to release and exonuclease or protease it only makes sense to do this if you know it isn't going to be swept away in a stream (release molecule and have more of the same species of bacteria you know you are not free-floating, if release the molecule and you sense it you know you aren't free floating

is feast or famine more rare for bacteria?

Famine is common, feast is rare

what is one way that quroum sensing does not reflect bacteria in the natural enviornment?

Feast and famine is a way that QS doesn't reflect the natural bacteria in the environment

which gene in the lac operon are hard to mutate for constituative gene expression?

Hard to mutate lac z and y for constituative

cis vs. trans If a gene product affects only what is on the same piece of DNA how does it act? If a gene produce affects a target located anywhere in the cell how does it act? what does this mean in general? what kind of mutations make it hard to determine between cis and trans acting mutations?

If a gene product affects only what is on the same piece of DNA --> acts in cis If a gene produce affects a target located anywhere in the cell --> acts in trans In general: proteins that can diffuse act in trans (e.g., transcription factors) : target sites for proteins act in cis (e.g., promoter) NOTE: It is difficult to tell difference btw cis- and trans-acting dominant mutations. To do so requires knowing if wt copy is being expressed (fuse wt to reporter gene).

Q: Where would a constitutive mutation occur? (note: operon is on in absence of Trp)

If don't add Trp, operon is normally on!

Trade-off in control re: amount of LacI. example? what is the idea behind this?

If increase LacI, get greater inhibition of gene expression during repression, but won't get as much removal of repression during induction (when you have the inducer present). Example: If have 1000 LacI, 0.04% of lacO will be free during repression but only 40% of sites will be free during induction (in the presence of inducer). Idea is that all these things are interconnected Protein specificity and amount of protein and parameters the cell can control

So how does LacI work? IN SUMMARY How does the Lacl affect the affinity for O?

Inducer acts by lowering the Lac repressor specificity for the target site by ~1000X (but it can still bind DNA). This works because there are so few LacI molecules and so much DNA (so LacI-inducer rarely binds lacO. Inducer does not affect amount of LacI, it simply changes the LacI distribution on DNA. LacI (inducer) has 10^6 higher specificity for O than random DNA

Galactose (gal) operon is senstive to what? what is galactose used as? why do we need the operon? what is needed to Gal utilization?

Is catabolite sensitive. Galactose is used as energy source and for other things (so need operon to have a basal level of transcription even in presence of Glu) These three genes are NOT sufficient for Gal utilization (requires galU and gene for permease, both located elsewhere on chromosome). Galactose is used both as energy source and for other things (e.g. to make polysaccharides).

trp attenuation model what is the leader peptide? why is it relevant?

Leader peptide = short peptide translation from leader RNA sequences which are involved in transcriptional or translational attenuation. It's relevant here because the structure of the leader RNA determines whether a transcription terminator is formed.

what is the mechanism of attenuation? Evidence for existence of additional control mechanisms: what are they and explain each

Mechanism to terminate transcription before RNAP reaches first structural gene 1 - remove repressor then always on, so you expect constitutive but they found the level of expression differed based on it trp was present 2 - levels of the tRNA for trp, when there is a lot of the tRNA then transcription terminated (role in tRNA and transcription - interaction) increased trp tRNA means decreased translation 3 - translation is involved in the transcription of the trp operon 4 - mutations in the leader region eliminate all this regulation whatever the connection b/t translation and transcription is it involves trpL

ATTENUATION: what type of mechanism is this? what is another example of this type of mechanism? what gene operon undergoes attenuation?

Mechanism to terminate transcription before RNAP reaches first structural gene --> another example of translation affecting transcription trp operon undergoes attenuation (watch https://www.youtube.com/watch?v=RQrdY07JkFU )

Merodiploid of wt & Lac+ mutant to determine what? what are the mutations in the lac operon that matter? why are these considered dominant?

Mutations that matter lac z, y, I , and p - Have mutated op that makes it dominant and constituative Repressor cant bind when this is mutated Repressor can still bind to the WT copy and it cant bind to the op on the mutated, so this is why it is considered dominant

what are the different lac operon mutations and what are there phenotypes? what are there functions affected? what is the mutation anytime a binding site is affected? what about when what is affected is a gene that codes for a product or protein? what type of mutation the lac operon has can be determined how?

NOTE: Be clear on whether are complementing with wt operon or one carrying a mutation in a different gene (most likely lacZ). ALL YOU NEED TO KNOW ANY TIME A BINDING SITE IS AFFECTED IT IS CIS ACTING ANY TIME IT IS A GENE THAT CODES FOR A PRODUCT/POLY PEP IT IS TRANS ACTING Summary of the lac operon and the type of mutations (can identify them by using complementation tests)

Sigma factors normal weight? what is the major sigma factor? how much do rpoH products weigh? How better to control expression of various genes than to encode a sigma factor specific for those genes? what does this imply? what does s32 control? what are hsp genes also involed in and what does this mean that they have? why does this make sense?

Normal sigma factor weight 70 kDa (s70 , encoded by rpoD gene) - the major sigma factor rpoH products weighs 32 kDa (s32) How better to control expression of various genes than to encode a sigma factor specific for those genes? This implies you have different kinds of promoters because diff sigma factors are going to recognize the same seq s32 controls hsp genes. Note: because hsp genes are also involved in non-stress responses..... .....most have two promoters (most have a weak s70 and strong s32) This makes sense because you have to make a lot more of a product during heat shock (sigma 70)

describe the lac operon regulatory region.... how strong is the promoter? why? describe the binding of lacl....what does it bind to? what is special about this binding site? what may lacl and RNAP bind to?

Not showing much of a structural gene, just the promoter site and things like that (+1) Lac op doesn't have a perfect -10 which means it isn't a perfect strong promoter The repressor binding site doesn't overlap with the RNAP binding site CAP = the binding site for a transcription activator and is located upstream (whatever is activating recruits RNAP) and the repressor generally blocks RNAP and binds closer to the binding site Lac has 3 operators and each o has subscripts LacI and RNAP may bind to different regions.

of the lac operon, for complementation tests which how many of the 5 total players are what? what happens if you mutate the operator? mutate the lacl? therefore what do you mutate?

Of the 5 players 3 are genes and 2 are target sites If you mutate the operator this would generate a constitutive cell because you alter the binding site for the repressor If you mutate lacI then you are mutating the repressor making a constitutive phenotype Therefore you mutate the promoter, the promoter is also smaller in size which is why It mutates much more frequently Mutating the promoter I think will generate a non functional lac z and t

can more than one type of attentuation occur? what is the common theme?

Other types of attenuation can occur, but common theme is whether a particular RNA structure if formed

Galactose (gal) operon how is PG1 activated and what does PG2 not require?

PG1 is activated by cAMP-CAP PG2 does not require CAP (cAMP-CAP represses this promoter) These three genes are NOT sufficient for Gal utilization (requires galU and gene for permease, both located elsewhere on chromosome). Galactose is used both as energy source and for other things (e.g. to make polysaccharides).

what is occurring when RNA pol pauses at the 1:2 pause site? what occurs if Trp is present? what does it affect?

Pol is transcribing and region 12 hairpin Ribosome is loaded onto the nascent RNA before region 3 is transcribed If trp is present the ribo blows past the trp codons and goes to the stop You are blocking region one but also 32 if trp is present If the ribosome is blocking just region one or region 2 depends on what hairpin is formed Blocking region 1 only means 2 can bind with 3 If 1 and 2 are blocked means 34 hairpin is formed Trp only affects where the ribosome stalls! --> determines what hairpin forms and what regions it affects The hairpin will form as long as nothing is preventing it from forming (no ribosome means 12 pairs and 34 pairs)

what is the purpose of a complementation test? what are the different outcomes? what does the cell need to make histidine in regards to this?

Purpose: to determine if two mutations are in same gene To make histidine the cell needs 2 genes

trp attenuation model Q: What affects which of these two structures is formed? Q: what would happen if mutate DNA so region 2:3 cannot form? (=trpL75) Q: what would you expect if start codon was changed to AUA (trp29) such that translation doesn't start? Q: the cell is starved for another amino acid. What is the affect on trp operon?

Q: what would happen if mutate DNA so region 2:3 cannot form? (=trpL75) region 34 can form and then there is termination (noninducible) Q: what would you expect if start codon was changed to AUA (trp29) such that translation doesn't start? 34 wouldn't form Q: the cell is starved for another amino acid. What is the affect on trp operon? depends on what amino acid and where it is located. If starved for another amino acid (lucine) and there is not lucine tRNA that is available then the ribo will stall and will not keep moving. as long as it affects only region 1 and not region 2 then it is constitutive if it is the one before the stop and affects region 2 then 34 forms Makes sense there are 2 trp codons because if you start running low you can still stall at one of the 2 regions (1 or 2)

Coordinated gene regulation includes: describe each and what each does......can they ever be both?

REGULONS = many distinct genes whose expression is controlled by a regulatory molecule. diff genes controlled by the same reg molecule STIMULON = collection of genes turn on by same environmental signal genes turned on by a certain signal (increase in temp, and all the genes affected by this. Are controlled by same regulatory molecule? - then also regulon)

what are the Types of Transcription Factors? describe them and provide examples of each

Repressor proteins - bind to a site near the promoter called operator (usually short and palindromic) to prevent transcription (e.g., by preventing RNAP binding or preventing it from movement)after it binds - Ex: LacI Activator proteins - binding of activator to DNA makes it easier for RNAP to bind promoter. - Ex: CAP

So how does LacI work? how many Lacl molecules are there per cell? how many repressor molecules? what does the inducer do? why does this work? what does the inducer not affect? what does it actually do?

There are ~ 10 LacI molecules per cell. (only about 10 repressor molecules per cell) Inducer acts by lowering the Lac repressor specificity for the target site by ~1000X (but it can still bind DNA). - just recognizes the op 1000X less efficiently when an inducer is present than when it isn't. normally has 1 million fold but add the inducer and it drops to 1000 fold This works because there are so few LacI molecules and so much DNA (so LacI-inducer rarely binds lacO. (10 repressors and 4.5 million base pairs in E.coli) Inducer does not affect amount of LacI, it simply changes the LacI distribution on DNA. (changes where it is bound)

what is a Complementation Test used to determine? how does this work? what does this assay assume?

To determine if mutations are in same or different gene - put two mutations in same cell --> restore wt phenotype? - as learned previously They want to determine what the phenotype will be when the 2 genes are mutated This assay assumes the gene product is diffusible and can move (i.e. acts "in trans").

TABLE OF OPERON CHARACTERISTICS for lac, gal and trp +/- regulation? Inducer/corep = ? Promoters? Operators? Repressors? Catabolite Sensitive? Repressor regulated? Autoregulated?

Trp is making (building things up) and even though it is negative it can still be repressible

trp operon regulation - model what is the aporepressor? what does it do and what is the corepressor? what occurs in the absence and presence of tryptophan?

TrpR is aporepressor (= repressor of neg. regulated operon) TrpR binds as dimer Trp is corepressor

heat shock proteins system? response? regulatory gene(s) and protein(s)? category of mechanism? some genes, operons, regulons and stimulons? what are the categories of hsps? what do chaperones do?

Two general categories: of Hsps 1) proteases (Lon, Clp) 2) chaperones (GroEL, DnaK/J) Chaperones - help proteins properly fold

How to isolate a constitutive mutation in trp operon what type of selection is this?

Use 5-methyltroptophan. --> 5-MeTrp acts as corepressor but cannot be used to make proteins --> Therefore, 5-MeTrp will lead to cell starvation unless constitutive mutant (example of positive selection)

When E. coli is grown in absence of lactose, there are how many beta-gal molecules per cell? what happens when lactose is added? what about when the lactose is removed? what is this an example of? what is the purpose of beta-gal and why is it so important?

When E. coli is grown in absence of lactose, there are ~5 beta-gal molecules per cell. When lactose is added, more enzyme is made within minutes (can comprise 10% of total soluble proteins). When lactose is removed, enzyme synthesis stops rapidly (within minutes). a type of control Beta gal can make the inducer that is important (allolactose which is the inducer) --> the enzyme that breaks it down The sugar molecule isn't the inducer, the break down molecule is and this is why you need beta gal because this makes the inducer needed to break down lactose

what is the Most efficient point in the central dogma to change the phenotype?

at the transcription level

Tryptophan (trp) operon is what type of operon? what types of regulation does it have? what happens when Trp (corepressor) is present?

biosynthetic operon (makes Trp), negative/repressible Three types of regulation Repressor is TrpR (trpR is unlinked to trp operson) Corepressor = Trp (is produce of trp operon) When Trp (corepressor) is present, no need to make more Trp (i.e. no gene expr is desired when Trp is present).

titration experiment what did it confirm? how was this experiment performed?

confirmed two operators

Q: What happens when glucose is added to medium that contains lactose? is this advantageous?

coordinating gene expression A: Two things - enzymes used to metabolize glucose are turn on - enzymes used to metabolize lactose (non-glucose) are turned off Cell doesn't need to worry about lactose because lactose broken down is glucose, so they will just deal with glucose when both are present It is advantageous for the cell to be able to turn off the lactose enzymes (ability of control)

what is QUORUM SENSING? what does it use? how is QS used? what are the chemical signals for QS?

density-dependent mechanism by which bacteria coordinate expression of specific target genes in response to critical concentration of signal molecule. uses two-component signal transduction How is QS used? 1) coordinate attack 2) synchronize cellular functions (Ex: root nodulation) 3) diffusion sensing Chemical signals 1) AHL (N-acylhomoserine lactones), used by Gram (-) = AI-1 (autoinducer) 2) peptide signals (AIPs = autoinducer peptides), used by Gram (+) 3) AI-2, used by both G(-) and G(+); produced by LuxS synthase 4) additional signals Ex: AI-3, DSF (diffusible signal factor), PSQ (Pseudomonas quinolone signal)

The Lac Operon - overview VIDEO

https://www.youtube.com/watch?v=2j7_2maQnng

Jacob and Monod what did they know? what did they make and what categories did they fall under? what was the step taken after this? why?

knew lactose induced production of b-galatosidase made mutants which fell into two categories: Lac+ and Lac- Next step = pairwise complementation tests (to determine how many genes there are)

Regulation Mechanisms of Specific Genes starts with what operon? what happens in the presence and absence of the substance?

lac operon LacI gene - P lac (which means promotor of lac) In the absence of lactose the lacI binds the op and you don't get transcription In the presence of lactose then it is broken down into allolactose and binds the lac repressor and therefore no longer prevents RNA pol from binding (neg)

REGULON in signal transduction example? Q: How do external signals get inside the cell? what is an example of this?

signal transduction (another example other than heat shock) -process of transmitting extracellular information (osmotic pressure, [cell], nitrogen availability) to inside the cell A: Some enter directly (e.g., metabolites) - these have to be small and still have to pass through a lot of things (membranes etc), other environmental changes are recognized by membrane proteins. --> gives rise to system to transmit external environment change to change in gene expression One example = two-component regulatory system

trp attenuation model where does this occur? where is the termination terminator? what different complexes can form and what happens when you get them? what does attenuation allow to form?

w/in the leader region, there are codons 2 codons are adjacent and both are for trp (if trp present you want to stop transcription) 34 = transcription terminator - get this when trp is around If 23 forms then region 3 can complex with 4 and oyu don't get transcription termination - you will have this when there is no trp around These hairpins cant form together (only get one) - 23 only forms if something prevents 12 from forming and this is based on if there is something at the trp codons (if something stalls there is it over region one and if region one isn't available then you make the 23 hairpin) Attenation can allows an intrinsic terminator to form