test 3 chapter 10

Constitutive Mutations pt 2

•A rarer constitutive mutation is cis-acting allowing constitutive expression of the lacZ and lacY genes even in the presence of a wild type copy of the lac DNA •These were named lacOC mutations for lac operator-constitutive mutations lacOC mutations cannot be complemented by a WT lacO region in the chromosome and are therefore cis-acting (cis dominant)

Attenuation of Transcription

•Activators and repressors turn transcription from the promoter on and off •Attenuation allows transcription to begin at the promoter but terminates it before the RNA polymerase reaches the first structural gene •Attenuation only occurs if the products of the operon are not needed •The classic example of attenuation in E. coli is the trp operon

Autoregulation of AraC

•AraC regulates not only expression of the ara operon but also its own expression •When arabinose isn't present, AraC are bound at araO2 and araI1 bending the DNA in the region of the araC promoter pC •If the concentration of AraC becomes too high it binds to the operator araO1 preventing transcription from pC

Model for Attenuation

•Attenuation is based on which of several secondary structure hairpins forms in the leader RNA •Which hairpin forms is determined by the levels of aminoacylated tRNATrp •The trpL region contains two trp codons which serve as the signal allowing the ribosome to "test the water" before the RNA polymerase plunges into the structural genes of the operon •If levels of tryptophan are low the levels of tRNATrp will also be low •When the ribosome encounters the trp codon it stalls, unable to insert a tryptophan •This stalled ribosome in the trpL region signals that tryptophan concentration is low and transcription should continue

Regulation of Gene Expression

•Cells need a way to express only the genes that it needs in a particular environment (saves energy) •Cells may need to turn off genes that may whose products may interfere with other processes at a given time •Many opportunities for regulation: -Transcriptional regulation -Post-transcriptional regulation (example: translational) •TRANSCRIPTIONAL REGULATION -Most transcriptional regulation occurs at the promoter through proteins called transcriptional regulators -Regulation of transcription can be negative, positive, or both -NEGATIVE REGULATION - expression is controlled by a REPRESSOR that binds to an operator sequence in the DNA and prevents initiation of transcription -POSITIVE REGULATION - transcription is controlled by an ACTIVATOR that is required for initiation of transcription by RNA polymerase -Some regulators can either be repressors or activators depending on where they bind

Complementation Tests

•Complementation tests require the cells to be partial diploid •Jacob and Monod utilized prime factors •To determine if a particular lac mutation was recessive or dominant they introduced an F′ factor carrying the WT lac region into a strain with the lac mutation in the chromosome •If the partial diploids are Lac+ the lac mutation is recessive •If the partial diploids are Lac- the lac mutation is dominant •They discovered most mutations were recessive so presumably inactivate genes whose products are required for lactose utilization •To determine how many genes are represented by recessive lac mutations they performed pairwise complementation tests between the different mutants •If the partial diploids are Lac+, the 2 recessive mutations complement each other and are therefore in different genes; Lac- = same gene •They found that most lac mutations sorted into two different genes which they named lacZ and lacY

Incorrect Prediction

•Constitutive araCC mutations should be dominant over wild-type allele in complementation tests •If AraC acts solely as an activator, partial diploid cells that have both an araCC allele and a wild type allele would be expected to constitutively express the operon (should be dominant over wild type) •Test it by introducing an F′ prime factor carrying a wild-type ara operon into a cell with an araCC •Prediction is that the genes will be constitutive

Isolating Constitutive Mutants of ara Operon

•Constitutive mutants of the ara operon are so rare they require special "tricks" to isolate them: • •One uses the anti-inducer D-fucose - binds to the AraC protein and prevents it from binding L-arabinose thus preventing operon induction •Wild-type E. coli cannot multiply to form colonies on plates containing D-fucose with L-arabinose as the sole carbon source •Only mutants which constitutively express the genes of the ara operon can form colonies under these conditions

Isolating trpR Mutants

•Constitutive mutations in trp operon are common and usually map in trpR •Constitutive mutants can be selected for by selecting for mutants resistant to the tryptophan analog 5-methyltryptophan •This analog binds to the TrpR repressor and acts like a corepressor but it cannot be used to make proteins like tryptophan can •This causes the trp operon is not induced even though tryptophan is absent and the cells will starve for the amino acid •Only constitutive mutants that express the operon in the presence of 5-methyltryptophan can multiply to form colonies on plates containing the analog but lacking tryptophan •The trp operon is also subject to a different type of regulation called attenuation (discussed later) •The first enzyme in the pathway is subject to feedback inhibition by the last enzyme in the pathway (discussed later)

Evidence for Positive and Negative Regulation

•Differences when regulatory gene is inactivated: -Negatively regulated operon - mutational inactivation of the regulatory gene allows transcription of the operon genes, even in the absence of inducer -Positively regulated operon - mutational inactivation of the regulatory gene prevents transcription of the genes of the operon even in the presence of inducer •Constitutive mutant - a mutant in which the genes of an operon are always transcribed even in the absence of inducer (more common with negatively that positively regulated operons) •Differences during complementation tests: -Constitutive mutations to a negatively regulated operon are often recessive to the wild type (repressor encoded by a wild-type copy can complement the mutated repressor) -Constitutive mutations to a positively regulated operon are often dominant to the wild type (a mutant activator protein that is active without inducer might activate transcription even in the presence of a wild-type activator protein)

Early Evidence for Positive Regulation

•Early evidence indicated the lac and ara operons are regulated by very different mechanisms •Loss of the regulators results in very different phenotypes: -Deletions and nonsense mutations in araC lead to a "superrepressed" phenotype in which the genes are not expressed even in the presence of the inducer arabinose -Mutations in the regulator for negatively regulated operon (lac) results in a constitutive phenotype •The frequency of constitutive mutants is another difference: -Mutants that constitutively express a negatively regulated operon are common (any mutation that inactivates the repressor gene causes constitutive expression) -Mutants that constitutively express the ara operon are extremely rare

Involvement of the Leader Region trpL

•Evidence suggested that this type of regulation targeted not the promoter but a region downstream of the promoter called the leader region or trpL •Deletion of the leader region eliminates regulation by attenuation •Double mutants with deletions of trpL and a trpR mutation are completely constitutive for expression of the trp operon •Deletions of trpL were found to be cis-acting affecting only expression of the trp operon on the same DNA •Transcription of the operon was found to terminate in the trpL region in the presence of tryptophan because of excess tRNATrp

Posttranslational Regulation: Feedback Inhibition

•Feedback inhibition - the end product of the pathway binds to the first enzyme of the pathway inhibiting its activity •Common in many biosynthetic pathways •A more sensitive and rapid mechanism for modulating the amount of the end product than are transcriptional regulation and translational regulation The trp operon uses feedback inhibition

Hairpin Formation

•Four different regions in the trpL leader RNA, regions 1, 2, 3, and 4 can form three different hairpins, 1:2, 2:3, and 3:4 •Formation of hairpin 3:4 causes RNA polymerase to terminate transcription because this hairpin is part of a factor-independent transcription termination signal •After RNA polymerase initiates transcription at the promoter, it moves through the trpL region to a site located just after region 2, where it pauses •The hairpin formed by region 1 and region 2 is the signal to pause •The pause if very brief (1 second) but it gives a ribosome time to load onto the mRNA before the RNA polymerase proceeds to region 3 •Ribosome probably helps release the paused RNA polymerase by colliding with it

Feedback Inhibition of the Isoleucine-Valine Operon

•If E. coli cells are presented with high concentrations of valine they will die as long as isoleucine is not provided in the medium •Valine and isoleucine are synthesized by the same pathway encoded by the ilv operon •The first enzyme of the pathway is feedback inhibited by valine, so the cells make neither valine or isoleucine The cells starve for isoleucine unless it is provided in the medium

trp Operon

•If TrpR was the only way to regulate expression of the trp operon, then levels of the enzymes in a TrpR mutant should always be the same whether tryptophan is present or absent •However, in a trpR null mutant, expression of the enzymes is higher when tryptophan is absent than when its present

Hairpin 3:4 = Transcription Terminates

•If the ribosome does not stall at the trp codons, it will continue until it reaches the UGA stop codon at the end of the trpL •It remains there while region 4 is synthesized and it prevents formation of hairpin 2:3

Mutations of the lac Operon

•It was already known that the enzymes of lactose metabolism are inducible (only made when lactose is present) •To understand the regulation of the lactose genes, Jacob and Monod isolated many mutations affecting lactose metabolism and regulation •These fell into two groups: 1.Mutants unable to grow with lactose as the sole carbon source (Lac- mutants) 2.Mutants that made the lactose-metabolizing enzymes even in the absence of lactose (constitutive mutants) •Jacob and Monod needed to know which of the mutations affected trans-acting gene products and how many different genes were represented •They also needed to know if any of the mutations were cis-acting (affecting sites on the DNA) •How do you think they achieved this?

lacI Mutations

•LacI contains different domains with different functions (operator binding and tetramer formation) - mapping of the structure of the gene provided insight into the control of the operon •Position of missense mutations in LacI cause a particular phenotype and may reveal information about which regions of LacI are responsible for particular functions •Need to isolate numerous different types of lacI missense mutations and map them to determine their location

Involvement of tRNATrp

•Mutations in the tryptophanyl-tRNA synthetase, mutations in the structural gene for the tRNATrp, and mutations in genes whose products are responsible for modifying the tRNATrp all increase expression of the operon •All of these mutations would lower the amount of tRNATrp •Suggests that the operon is not sensing the amount of free tryptophan in the cell but, rather, the amount bound to the tRNATrp

Isolating Deletion Mutations of lacI

•One way to select lacI deletions is to select mutations in a nearby gene and screen them to identify those that are also lacI- (most of these are deletions that extend into lacI gene) •Investigators decided to use the tonB gene since mutations are easy to select for. How? •A subset of tonB mutations will be deletions and some will extend into lacI gene •Unfortunately, tonB is not near lacI (so it had to be placed there by integrating a prophage carrying the lacI gene) •tonB mutants are selected by plating on phage T1 and deletions extending into lacI are detected by plating on X-Gal •Constitutive lac mutants make blue colonies on X-Gal plates in the absence of inducer •Deletions must end in the lacI gene and not extend into lacZ •Endpoints of deletions in lacI gene were mapped by crossing them with a few point mutations that had been mapped previously by three factor crosses

cis-Acting lac Mutations

•Other lac mutations adjacent to the lacZ gene were much rarer and could not be complemented to allow expression of the lac genes on the same DNA even in the presence of good copies of the lac genes •These mutations were in cis acting affecting a site on the DNA rather than a diffusible gene product •To show the mutation is cis acting they introduced an F′ factor containing the potential cis-acting lac mutation into a cell with mutations in lacZ and lacY •Any trans-acting gene products encoded by the F′ factor lacZ or lacY genes would complement the chromosomal mutations •If the phenotypes are Lac- then the mutation in the F′ factor must prevent expression of both LacZ and LacY from the F′ factor and therefore be cis acting •They named one of the cis acting mutations "lacp mutations" (promoter)

Positive Regulation

•Positively regulated operons are under the control of an activator protein •Genes of these operons are only transcribed in the presence of the activator when the inducer is bound •Example: the L-ara operon responsible for utilization of the sugar L-arabinose • •L-ara operon consists of three structural genes: araA, araB, and araC which are transcribed from a single promoter pBAD •The activator protein AraC binds to a site upstream of the promoter called the araI region to activate transcription in the presence of the inducer arabinose •Also just upstream of the araI site is the CAP site where CAP binds •There are two operators, araO1 and araO2, at which the AraC protein binds to repress transcription •The araC gene is transcribed from a promoter pC in the opposite direction from pBAD

Constitutive Mutations pt 3

•Some lacI mutations, called lacI-d are trans "dominant" •These mutations make the cell constitutive for lac gene expression even in the presence of a good copy of the lacI gene •This is because the LacI repressor forms a homotetramer (4 polypeptides) •A mixture of normal and defective subunits can be nonfunctional causing the constitutive LacI- phenotype •Another type of mutation lacIRC changes the repressor so that it binds the operator only in the presence of inducer

Lac- Mutants with Dominant Mutations

•Some mutations affect diffusible products which are dominant rather than recessive •These mutations make the cell Lac- even if there is a good copy of the operon in the cell, either in the chromosome or the F′ factor •These dominant lac mutations are called lacIS mutations for "superrepressor mutations" What do you think is happening with this type of mutation??

Constitutive Mutations

•Some mutations do not make the cells Lac- but rather make them express lacZ and lacY genes even in the absence of inducer •These types of mutations were also found to be either dominant (non-inducible) or recessive (inducible) •Complementation between the recessive constitutive mutations revealed they are all in the same gene which they named lacI The lacI mutation can be complemented and so other genes on the prime factor will be inducible in the presence of a WT copy of the lacI gene (trans recessive)

An Early Model of the ara Operon

•The AraC protein can exist in two states: P1 and P2 -P1 - in the absence of the inducer (L-arabinose) AraC is inactive -P2 - in the presence of the inducer (L-arabinose) AraC is active - binds to the araI site in the promoter and activates transcription of the operon •Explains why mutations in araC that cause the constitutive phenotype are rare but do occur •araCC mutations change AraC so that it is permanently in the P2 state even in the absence of L-arabinose (Constitutive) •Such mutations would be very rare since only a few amino acid changes in the AraC protein could change the conformation to the P2 state

AraC Has Two Functions

•The P1 form of AraC in the absence of arabinose is not just inactive but takes on the role of an anti-activator •The AraC P1 state preferentially binds to the operator araO2 and another site araI1 bending the DNA •When bound to araO2 it cannot bind to araI2 and activate transcription from the pBAD promoter •In the presence of arabinose the AraC protein changes to the P2 state and now preferentially binds to araI1 and araI2 activating the operon •Explains why araCC mutations are recessive to wild type •AraCC in the P2 form can't bind araI1 and araI2 if the wild-type AraC is already bound to araO2 and araI1

Evidence for the Attenuation Model

•The existence and function of the 2:3 hairpin were supported by phenotypes produced by mutation trpL75 •This mutation changes one nucleotide and prevents formation of the 2:3 hairpin •In trpL75 mutants transcription terminates in the trpL region even in the absence of tryptophan • •Translation of the leader peptide is also essential since the ribosome at the stop codon is what prevents hairpin 2:3 from forming and subsequently allows hairpin 3:4 to form •Mutation trpL29 changes the AUG start codon of the leader peptide to AUA preventing translation initiation •In trpL29 mutants termination also occurs even in the absence of tryptophan •Without a ribosome stalled at the trp codons, hairpin 1:2 will persist and hairpin 3:4 will form terminating transcription

Negative Regulation of Biosynthetic Operons

•The genes of the lac operon work to degrade compounds to obtain catabolites in order to build other molecules (catabolic or degradative operons) •Some operons synthesize compounds needed by the cell such as amino acids, nucleotides, and vitamins (biosynthetic operons) •Biosynthetic operons should not be turned on when the end product of the pathway is available •If the genes of a biosynthetic operon are constitutively expressed in the absence of a regulatory gene product, the operon is negatively regulated •The effector that binds to the repressor and allows it to bind to the operators is called the corepressor •A repressor that negatively regulates a biosynthetic operon is NOT active in the absence of a corepressor and is in this state called a aporepressor

Jacob and Monod Operon Model

•The lac operon contains the structural gene lacZ and lacY which encode the enzymes required for utilization of lactose •lacZ gene product is a β-glactosidase that cleaves lactose to form glucose and galactose, which can then be used by other pathways •lacY gene product is a permease that allows lactose into the cell •lacA is another gene included in the operon that encodes a transacetylase whose function is unknown •The product of the lacI gene is repressor protein which binds to the operator sequence (lacO) close to the promoter preventing RNA polymerase from binding •When lactose is available, the inducer binds to the repressor and changes its conformation so that it can no longer bind the operator sequence •RNA polymerase can then bind to lacp and transcribe the lacZ, lacY, and lacA genes

Negative Regulation - The E. coli lac Operon

•The lac operon is the classic example of negative transcription regulation •The operon encodes the enzymes responsible for the utilization of the sugar lactose •Characterized by the studies of Jacob and Monod in the late 1950's and their model still serves as the framework by which all other studies of gene regulation are compared •It earned Jacob and Monod a Nobel Prize in 1965, and today it serves as the paradigm for understanding gene regulation in other organisms

Hairpin 2:3 = Transcription Continues

•The progress of the ribosome through the trp codons of the trpL determines whether hairpin 3:4 will form, causing termination, or whether region 2 will instead pair with region 3 •The 2:3 hairpin will form if the ribosome stalls at the trp codons because of low tryptophan concentrations

The trp Operon of E. coli

•The trp operon encodes enzymes required for the synthesis of L-tryptophan when none is available in the media •Five structural proteins are transcribed from a single promoter ptrp •The trp operon is negatively regulated by the TrpR repressor protein that is unlinked to the rest of the operon

Feedback Inhibition of the trp Operon

•The tryptophan analog 5-methyltryptophan, at high concentrations, binds to anthranilate synthetase and inhibits the activity of the enzyme •This starves the cells for tryptophan •Only mutants defective in feedback inhibition because of a mutation in the trpE gene that prevents the binding of tryptophan (and 5-methyltryptophan) to anthranilate synthetase enzyme can multiply to form a colony in the absence of tryptophan

Negative regulation of the trp operon

•TrpR can prevent transcription of the operon by binding to the promoter ptrp •TrpR can bind to the operator region only if the corepressor tryptophan is present in the medium •Tryptophan changes the conformation of TrpR aporepressor so that it can bind the promoter •TrpR also autoregulates its own expression