7.1-7.4

Structure of trp operon

- five genes code for the polypeptides in the enzymes needed for trp synthesis - the trp operator lies wholly within the trp promoter

Overall layout of lac operon

- the use of the L1 deletion proved that the CAP-cAMP activator-binding site is to the left of the promoter

Binding sites for CAP

Binding sites for CAP in lac, gal, and ara operons all contain the sequence TGTGA - sequence conservation suggests an important role in CAP binding - binding of CAP-cAMP complex to DNA is tight

The mechanism of CAP action: How do CAP and cAMP stimulate transcription?

The CAP-cAMP complex binds to the lac promoter - using mutants and mapping, found that the activator-binding site (CAP-cAMP site) lies just upstream of the promoter - binding of CAP and cAMP to the activator site helps RNA pol form an open promoter complex

The araCBAD operon

The ara operon is also called the araCBAD operon for its 4 genes - three genes araB, A, and D, encode the arabinose metabolizing enzymes (what we are wanting to express or repress) - these are transcribed rightward from the promoter araPBAD - other gene, araC, encodes the control protein AraC and is transcribed leftward from the araPC promoter

Repression and derepression of the lac operon

The repressor is an allosteric protein - binding of one molecule to the protein changes shape of a remote site on that protein, altering its interaction with a second molecule The inducer, allolactose (an alternative form of lactose) binds the repressor - causes the repressor to change conformation that favors release from the operator

Binding of the repressor

When the repressor binds to the operator, the operon is repressed. - this is because the operator and promoter sequence are contiguous (right next to each other) - when the repressor occupies the operator, it appears to prevent RNA pol from binding to the promoter and transcribing the operon - as long as no lactose is available, the lac operon is repressed

A combination of genetic and biochemical experiments revealed the two key elements of negative control of the lac operon are:

the operator and the repressor

Summary of CAP-cAMP action

- CAP-cAMP binding to the lac activator-binding site recruits RNA pol to the adjacent lac promoter to form a closed complex - this closed complex then converts to an open promoter complex - CAP-cAMP causes recruitment through protein-protein interaction with the αCTD of RNA pol - CAP-cAMP also bends its target DNA by about 100 degrees when it binds

7.4 Riboswitches

- small molecules can act directly on the 5'-UTRs (untranslated regions) of mRNAs to control their expression - regions of 5'-UTRs capable of altering their structures to control gene expression in response to ligand binding are called riboswitches - another example of allosteric control

Discovery of the operon

During the 1940s and 1950s, Jacob and Monod studied the inducibility of lactose metabolism in E. coli and found: - three enzyme activities/three genes were induced together by galactosides - constitutive mutants: those that need no induction, so produce the three gene products all of the time - created merodiploids: partial diploid bacteria carrying both wild-type (inducible; lacI) and constitutive (not inductible; lacI-) alleles - using merodiploids distinctions could be made by determining whether the mutation was dominant or recessive

ara operon summary

The ara operon is controlled by the AraC protein - represses by looping out the DNA between 2 sites, araO₂ and araI₁ that are 210 bp apart Arabinose can derepress the operon causing AraC to loosen its attachment to araO₂ and bind to araI₂ - this breaks the loop and allows transcription of operon CAP and cAMP stimulate transcription by binding to a site upstream of araI - AraC controls its own synthesis by binding to araO₁ and prevents leftward transcription of the araC gene

The mechanisms of repression

Two competing hypotheses of mechanism for repression of the lac operon: 1.) RNA pol and repressor can bind together to the lac promoter: repressor blocks the transition from initial abortive transcribing complex to elongation state. Pol-promoter complex is in equilibrium with free pol and promoter. (evidence favors this hypothesis) 2.) The repressor, by binding to the operator, blocks access by pol to adjacent promoter.

Types of ara operators

Two types exist: - araO₁: regulates transcription of a control gene called araC - araO₂: located far upstream of the promoter it controls (PBAD)

Riboswitch regions

- aptamer: region that binds the ligand and is right next to the... - expression-platform: another module in the riboswitch which can be a terminator, ribosome-binding site, or another RNA element that affects gene expression By binding to its aptamer and changing the conformation of the riboswitch, a ligand can affect the expression platform and thereby control expression.

Mechanism of attenuation

- attenuation imposes an extra level of control on an operon (extra control needed because repression of this operon is weaker than previous operons) - contains leader-attenuate whose purpose is to attenuate, or weaken, transcription of the operon while trp is abundant - operates by causing premature termination of the operon's transcript when product (trp) is abundant - the reason for this premature termination is that the attenuator contains a transcription stop signal (terminator) which is an inverted repeat that forms a hairpin - in the presence of low trp concentration, the RNA pol reads through attenuator so the structural genes are transcribed

Defeating attenuation

- attenuation operates in the E. coli trp operon as long as tryptophan is plentiful - if amino acid supply low, ribosome stalls at the tandem tryptophan codons in the trp leader - because the trp leader is still synthesized, the stalled ribosome will influence the way RNA folds - prevents formation of an appropriate hairpin (terminator, between region 3 and 4), allowing for RNA pol to transcribe the operon and form trp

Determining whether the mutation is dominant or recessive: cis-acting

- because an operator controls only the operon on the same DNA molecule it is called a cis-acting gene (affects operon on same DNA strand) - thus, a mutation in one of the operators in a merodiploid should render the operon on that DNA molecule unrepressable, but should not affect the operon on the other DNA molecule - we call such a mutation cis-dominant because it is dominant only with respect to genes on the same DNA - these mutations are called Oc for operator constitutive

Determining whether the mutation is dominant or recessive: trans-acting

- because the repressor gene produces a repressor protein that can diffuse throughout the nucleus, it can act on multiple DNA strands and bind to both operators in a meriploid (wild-type and constitutive) - we call such a gene trans-acting gene because it can act on loci on both DNA molecules in the merodiploid (affects both operons) - a mutation in one of the repressor genes will still leave the other repressor gene undamaged, so its wild-type product can still diffuse to both operators and turn them off -thus, such a mutation should be recessive

Activation of lac P1 transcription by CAP-cAMP experiment

- did run-off transcription assay to measure transcription with vs without cAMP-CAP, looking at P1 and UV5 (two different versions of lac operon promoters) - P1 promoter is the standard promoter for the lac operon and is CAP-cAMP dependent (inducible), so transcription was stimulated with CAP-cAMP - UV5 is a strong promoter that is cAMP-CAP independent so it is active all the time and cAMP-CAP did not stimulate transcription - when truncated alpha subunit lacking CTD, lost inducibility of P1 by cAMP-CAP (last two enzymes have truncated alpha subunits)

History of riboswitches

- early in the evolution of life it is hypothesized that genes were made of RNA, not DNA, and enzymes were made of RNA, not protein - without proteins to control their genes, life forms in the RNA world would have to rely on small molecules directly with their genes - if this hypothesis is true, riboswitches today are relics of the most ancient forms of genetic control

Experiments with cAMP

- experiments demonstrated that cAMP could be added to E. coli to overcome catabolite repression of the lac operon (and a number of other operons like gal and ara) - the addition of cAMP leads to the activation of the lac gene even in the presence of glucose

Glucose and lactose in E. coli

- glucose is the primary sugar that E. coli uses - when glucose is all used up, there is a lag period of about an hour where cells turn on the lac operon and begin to accumulate the enzymes they need to metabolize lactose - then cell begins utilizing lactose and growth resumes - graph shows diauxic growth

Effects of regulatory mutations: wild-type and mutated operator operons

- in mutated, no repressor binds and lac products are made in the absence of lactose - because only the operon connected to the mutant operator is affected, this mutation is cis-dominant

Positive control of the ara operon

- is also mediated by CAP and cAMP - the CAP-cAMP complex attaches to its binding site upstream of the araBAD promoter - DNA looping would allow CAP to contact the pol and thereby stimulates its binding to the promoter

Example of riboswitch action (ribD in B. subtilis)

- ligand (which can be a downstream product) binds to aptamer in riboswitch - binding of ligand causes base pairing in riboswitch to change to create terminator - transcription is attenuated which saves cell energy because genes are not expressed if product is not needed

Effects of regulatory mutations: wild-type and mutated repressor gene whose product cannot bind to operator

- mixtures of both wild-type and mutant repressor monomers still cannot bind to the operator - because the operon is derepressed even in the absence of lactose, the mutation is dominant - furthermore, because the mutant protein poisons the activity of the wild-type protein, we call the mutation dominant-negative (cannot bind to either operator in a merodiploid)

Effects of regulatory mutations: wild-type and mutated repressor operons

- mutation in repression gene resulting in no repressor being made - wild-type repressor gene makes enough normal repressor that repression of both operon occurs, so no lac products in absence of lactose - mutation is recessive

Effects of regulatory mutations: wild-type and mutated repressor gene that cannot bind the inducer

- mutation in repressor gene results in mutant repressor which joins with wild-type repressor and together they cannot bind inducer - the mutant repressor therefore binds irreversibly to both operators and renders both operons uninducible - keeps gene repressed and turned off even when inducer, allolactose, is present - no lac product in presence or absence of lactose - this mutation is therefore dominant

1.) Negative control: lac repressor

- product of the regulatory gene lacI - tetramer of 4 identical polypeptides - binds to the operator (where repressors bind at) just right of the promoter

Features of the ara operon

- the CAP-binding site is 200 bp upstream of the ara promoter, yet, CAP stimulates transcription - this operon has another system of negative regulation mediated by the AraC protein

7.3 The trp Operon

- the E. coli trp operon contains the genes for the enzymes the bacterium needs to make the amino acid tryptophan - the trp operon codes for anabolic enzymes, those that build up a substance - anabolic enzymes are typically turned off by a high level of the substance produced (negative control by repressor when trp levels are elevated) - trp operon also exhibits additional level of control called attenuation

7.2 The ara operon

- the ara operon of E. coli codes for enzymes required to metabolize the sugar arabinose - it is another catabolite-repressible operon (cAMP and CAP used)

How does araO₂ control transcription?

- the araO₂ operator controls transcription from a promoter 250 downstream - the DNA between the operator and promoter loops out - AraC proteins on O₂ and I₁ (bound to the same side of DNA) bind to each other - we know that there is a loop because when added multiples of 10 bp into the DNA, the operon still worked. With any different number, proteins could not bind in loop and operon did not work

Catabolite repression

- the ideal positive controller of the lac operon would be a substance that sensed the lack of glucose and responded by activating the lac promoter so that RNA pol could bind and transcribe lac genes - one substance that responds to glucose concentration is a nucleotide called cyclic-AMP (cAMP) - cAMP concentration rises as the concentration of glucose drops

Repressor-operator interactions experiment

- using a filter-binding assay, Cohn and colleagues found that the lac repressor binds to the lac operator - this experiment showed that a genetically constitutive lac operator has lower than normal affinity for the lac repressor, demonstrating that the sites defined both genetically and biochemically as the operator are in fact the same

Negative control of the trp operon

- without trp no trp repressor exists, just the inactive protein, aporepressor - if aporepressor binds trp, aporepressor changes conformation (allosteric transition) forming the trp repressor which has a high affinity for the trp operator - trp is a corepressor

Parts of the positive controller of the lac operon

1.) cAMP 2.) a protein factor known as: - catabolite activator protein (CAP) - gene encoding this protein is crp (cyclic-AMP receptor protein) Together they stimulate transcription.

Control of the lac operon

All three genes are turned on and off together, and are tightly controlled using 2 types of control: 1.) negative control: indicates that the operon is turned on unless something intervenes to stop it. This is done by the lac repressor which keeps the lac operon from being expressed in the absence of lactose. 2.) positive control: an activator, additional positive factor (CAP-cAMP), responds to low glucose by stimulating transcription of the lac operon. Keeps the operon relatively inactive in the presence of glucose. High glucose levels keep the concentration of the activator low (prevents waste of lactose because glucose is easier to metabolize).

B-galactosidase activity experiment

CAP-cAMP complex positively controls the activity of β-galactosidase - CAP binds cAMP tightly which led to high production of B-gal - mutant CAP does not bind cAMP tightly, so lower activity and production

Recruitment

CAP-cAMP recruits pol to the promoter in 2 steps: 1.) formation of the closed promoter complex 2.) conversion of the closed promoter complex into the open promoter complex - CAP-cAMP bends its target DNA by 100° when it binds (may help to melt DNA, separate strands, and form open complex) - in image R is RNA pol and P is promoter

2.) Positive control of the lac operon

Catabolite repression of lac operon - when glucose is present, the lac operon is in a relatively inactive state - this selection in favor of glucose metabolism and against the use of other energy sources has been attributed to the influence of some breakdown product, or catabolite, of glucose - it is therefore known as catabolite repression

Trp role in the trp operon

Functions in negative control - high trp concentration is the signal to turn off the operon because the presence of trp helps the trp repressor bind to its operator

CAP-cAMP activated operon promoters

Have very weak promoters. - their -35 boxes are quite unlike the consensus sequence - if these promoters were strong they could be activated even when glucose is present (RNA pol could form open promoter complexes readily without the help from CAP or cAMP; has to be weak to be dependent on CAP-cAMP)

AraC control of the ara operon

In absence of arabinose, no araBAD products are needed, AraC exerts negative control: - araC makes control proteins (packmans) which binds to araO₂ and araI₁ - this loops out the DNA in between araO₂ and araI₁ and represses the operon because pol cannot get to PBAD promoter In presence of arabinose, arabinose binds to AraC proteins, causing them to change conformation: - it can no longer bind araO₂ - occupies araI₁ and araI₂ instead - repression loop broken - operon is derepressed

7.1 The lac Operon

It was the operon (set of genes under the control of a promoter) discovered. It contains 3 genes (one promoter) coding for E. coli proteins that permit the bacteria to use the sugar lactose. 3 enzymes: - galactoside permease (coded for by the gene lacY): transports lactose into the cell - β-galactosidase (gene lacZ): breaks the lactose down into its monosaccharides- galactose and glucose - galactoside transacetylase (gene lacA): unclear function

Riboswitch action

Operates by depressing (reducing) gene expression: - some work at the transcriptional level - others can function at the translational level (more likely to be influenced by change in ribosome-binding site)

Overall negative control of the lac operon

Repression - when no lactose present and want to have operon turned off, lacI gene produces repressor monomer - four repressor monomers come together to form tetramer which binds to the operator and blocks RNA pol from transcribing the lac genes Derepression - lactose has to be present, particularly the inducer allolactose - allolactose binds to the repressor and causes it to change shape, removing it from the operator and allowing RNA pol to transcribe genes - this produces a polycistronic mRNA that is translated to B-galactosidase, permease, and transacetylase

The ara control protein

The AraC acts as both a positive and negative regulator. There are 3/4 binding sites: 1.) Far upstream site araO₂ 2.) araO₁ located between -106 and -144 3.) araI which has 2 half-sites: araI₁ between -56 and -78 and araI₂ -35 to -51. Each half-site can bind one monomer of AraC.

Types of lac operators

There are three: - the major lac operator lies adjacent to promoter - two auxillary lac operators: one upstream and other downstream All three are required for optimum repression. The major operator produces only modest amount of repression.

CAP plus cAMP allow...

formation of an open promoter complex - the role of cAMP is to change the shape of CAP to increase affinity for the activator-binding site - Unless CAP-cAMP complex is bound, the open promoter complex does not form even if RNA pol has bound the DNA

Genes of the lac operon

lacZ, lacY, and lacA are grouped and transcribed together producing 1 mRNA called a a polycistronic message (message with information from more than one gene) starting from a single promoter. - each cistron, or gene, has its own ribosome binding site - this allows each cistron to be translated by separate ribosomes that bind independently of each other

Proposed CAP-cAMP activation of lac transcription

the CAP-cAMP dimer binds to the activator site on the DNA and simultaneously binds to the carboxyl-terminal domain of the polymerase α-subunit (α-CTD), facilitating binding of pol to promoter - this enhances binding between promoter and pol