Chapter 17 - Regulation of Gene Expression in Bacteria and Bacteriophages

lysogen induction

-SOS response of E. coli cells to environmental stress (mutagens, radiation) includes recA gene, protein involved in recombination repair of DNA -RecA become co-protease that stimulates latent protease activity in repressor, cleaving itself in half and releasing from operators -transcription begins from PR and PL, transcribing cro, which shuts down transcription of repressor gene -lysogeny is broken and lysis begins

x-gal/IPTG

-X-gal can act as substrate; when metabolized, turns blue -IPTG acts as an inducer

tryptophan starved

-as RNA polymerase is transcribing, ribosome is translating -when ribosome reaches trp codons in region 1, stalls bc trp is low; allows region 2 to pair with region 3, and region 4 is unpaired -RNA polymerase can continue past the attenuator and transcribe the structural genes (antitermination)

PlacI mutation

-because promoter for lacI is a relatively weak promoter, up mutations are possible, which will make the RNA polymerase bind with greater affinity, increasing the amount of repressor produced -lacIQ (quantity) and lacISQ (super quantity) -these mutations reduce the efficiency of induction (can still be induced at high lactose concentrations)

lysogenic

-cI gene has 2 promoters: PRM (repressor maintenance; requires its own pdt to activate) and PRE (repressor establishment, used initially) -PRE lies to right of both PR and cro; directs transcription leftward through cro and then through cI -PRE cannot be transcribed by E. coli RNA polymerase without cII (and cIII) gene pdt; helps polymerase bind to promoter sequence -sense cI transcript gives repressor, antisense transcript of cro probably hybridizes to sense transcript of cro to prevent its translation -repressor binds to operators OR and OL: turns off early transcription (interrupting lytic cycle, importantly cro), and encourages repressor synthesis by activating PRM -OR controls leftward transcription of cI and rightward transcription of cro; has 3 binding sites with different affinities for repressor: OR1>OR2>OR3; OR1 and OR2 binding is cooperative -repressor dimers bind to OR1 and OR2, very close to binding site for RNA pol at PRM - they touch which allows for more efficient initiation of transcription -binding at OR1 and OR2 prevents transcription from PRE because repressors block cII and cIII transcription; not a problem as long as OR3 is left open so RNA pol can transcribe cI from PRM -repressor may build up and bind to OR3, preventing any more transcription until repressor level drops again and dissociates from OR3, allowing cI transcription to begin again

positive regulation of lac operon

-catabolite activator protein (CAP, dimer) binds with 4 cAMP to form CAP-cAMP complex -complex binds to CAP site, upstream of where RNA polymerase binds to promoter -CAP recruits RNA polymerase to promoter, and transcription is initiated -in presence of glucose and lactose, glucose is used preferentially because of catabolite repression: glucose causes amount of cAMP to be greatly reduced, insufficient CAP-cAMP complexes form and transcription lowers, even though repressors are removed

effectors/effector molecules

-class of small molecules that help control the expression of regulated genes -includes inducers -allolactase and tryptophan

polycistronic mRNA

-contains information from multiple genes that are adjacent to one another and encode proteins that work together

attenuation

-controls how much trp is produced under trp starvation or trp limitation -reduces transcription by a factor of about 10

tryptophan nonstarved

-enough tryptophan is present so that the ribosome does not stall in region 1, stops at stop codon in region 2; allows region 3 to pair to region 4, causing polymerase to slow down -string of Us makes base pairing between DNA template and new RNA weak, polymerase falls off and RNA dissociates (rho independent termination)

temporal expression of genes

-expression of genes in a specific order or at a particular time -T7 phage: uses host RNA pol to synthesize its own phage-specific pol that transcribes remaining genes -phage SPO1: produces different σ factors at different stages of transcription to change specificity of host RNA pol

phage SPO1

-first 5 minutes: host RNA pol handles transcription of early SPO1 genes; produces gp28, which associates with host core pol and displaces host σ -next 5 minutes: RNA pol changes specificity; begins transcribing middle phase genes; produces gp33 and gp44, new σ factors that change specificity of RNA pol to the late phase genes

repressible operons

-gene activity is repressed when the end product is available (opposite of lac operon: activated by initial compound) -used commonly in anabolic (biosynthetic) pathways

constitutive genes

-genes whose products are essential to the normal functioning of a growing and dividing cell and are always on, no matter what the conditions are -ex: genes that code for enzymes needed for protein synthesis as glucose metabolism

immediate early

-host RNA polymerase holoenzyme transcribes immediate early genes (cro and N) which lie immediately downstream from rightward and leftward promoters, PR and PL -polymerase reaches end of immediate early genes, ρ dependent termination stops short of delayed early genes -cro gene product, Cro protein, is an antirepressor that blocks transcription of repressor gene cI, preventing synthesis of repressor protein (necessary if other phage genes are to be expressed) -N gene product, pN, is an antiterminator protein that coordinates NusA, NusB, NusE, and NusG (from host) to form antitermination complex that acts on RNA pol at nut sites; delayed early phase begins

fate of infection

-if cI wins, lysogeny occurs -if cro wins, lysis occurs -Cro binds to both OR and OL as does repressor, but order of binding affinity is opposite of that of repressor: OR3>OR2>OR1 -when Cro binds to OR3, cI transcription from PRM cannot occur (Cro acts as repressor) -when Cro fills up rightward and leftward operators, prevents transcription of early genes from PR and PL, including cII and cIII, so PRE cannot function, and repressor synthesis ceases, shifting to lytic cycle (turning off early transcription is also required for lytic growth; continued production of delayed early proteins late in infection can abort lytic cycle) -concentration of CII is important in determining whether cI or cro wins: under good environmental conditions, high proteases overwhelm CIII and destroy CII, ensuring lytic pathway; under starvation condition, protease levels are depressed, favoring lysogeny; makes sense because lytic pathway requires considerable energy

metabolism of lactose in E. coli

-in absence of lactose, lactose metabolism genes or inactive -in presence of lactose, three proteins are synthesized: 1) β-galactosidase: breaks down lactose into glucose and galactose, as well as catalyzing isomerization of lactose to allolactose (important compound in regulation of lac operon) 2) lactase permeases (M protein): found in cytoplasmic membrane, actively transports lactose into the cell 3) β-galactosidase transacetylase: transfers acetyl group from acetyl-CoA to β-galactosidase (function is not understood) -in presence of glucose, only ~3 molecules of β-galactosidase present in cell -in presence of lactose and absence of glucose, amount increases a thousandfold (coordinate induction)

phage λ

-infects E. coli and can either infect it (lytic mode) or integrate into host genome (lysogenic mode) -genome is linear; circularizes upon injection into E. coli cell due to "sticky ends" (cos); brings together late genes, which had been separated at two ends of linear genome

structural lac genes

-lacZ = β-galactosidase, lacY = permease, lacA = β-galactosidase transacetylase -genes are tightly linked in order lacZ-lacY-lacA -transcribed onto single polycistronic mRNA

late

-late genes all transcribed in rightward direction using late promoter PR' (downstream from Q); code fore proteins that make up phage head and tail, and for proteins that lyse the host cell -transcription from PR' terminates after only 194 bases, unless pQ prevents termination (pN will not work)

lacI-d mutation

-makes defective repressor subunits -constitutive -trans dominant: defective subunits and normal subunit form nonfunctional tetramer

lacOc mutations

-mutation in operator means repressor cannot bind -genes downstream of operator on same strand are constitutively expressed (cis dominant)

lacI- mutation

-mutation in repressor, cannot bind to operator -genes are constitutively expressed -in partial diploid, lacI+ is trans dominant to lacI- -lacI+ can make enough gene product to bind to both operators

mutations in attenuation

-mutations in region 3 or region 4 disrupt pairing so the structure is less stable; less able to prevent transcription -change trp codons to another amino acid: attenuation in response to changing levels of that amino acid

lacO gene

-operator, immediately upstream of lacZ -location where repressor binds to prevent transcription -cis dominant: only affects genes downstream from it on the same DNA molecule

lacZ- lacY+ / lacZ+ lacY-

-partial diploid test -example: lacI+ lacO+ lacZ- lacY+ / lacI- lacO+ lacZ+ lacY- -no inducer: sufficient repressors from lacI+ bind to both operators, no transcription -with inducer: repressor inactivated, both operons transcribed; one produces defective β-galactosidase and normal permease, one produces defective permease and normal β-galactosidase -both operons are under inducible control

Plac- mutation

-promoter mutation, structural enzymes are not produced, or are produces at low rates -cis dominant

trp operon

-promoter, operator, leader region (trpL, attenuator), structural genes (trpE, trpD, trpC, trpB, trpA) -regulated by trpR gene

lacI gene

-repressor gene, upstream of lacO -transcribed constitutively, 4 molecules form tetramer (Lac repressor) -has weak promoter, so few repressors are found -repressor binds to operator, overlaps sequence recognized by RNA polymerase, transcription does not occur (negative control) -allolactose binds to Lac repressor causing allosteric shift (can no longer bind to operator)

leaky expression

-repressors bind and dissociate, allowing some opportunity for RNA polymerase to bind to promoter and initiate transcription -important: this is why there are a few molecules of the three enzymes, which are necessary to transport lactose into the cell and convert it to allolactose to induce expression

coordinate induction

-simultaneous transcription of two or more genes brought about by an inducer

lacIs mutation

-superrepressor mutation, can bind to operator but cannot bind to allolactose, so operons are never transcribed -can never use lactose as carbon source -trans dominant

phage T7

-three phases of transcription: early, middle, and late -early: host RNA pol and host σ factor used to transcribe 5 class I genes; gene 1 is phage-specific RNA pol -RNA pol reads class II and III genes -very specific: only class II and III genes and nothing else -has a very strong promoter sequence (~200 nt): any change is a strong down mutation

inducible genes

-transcribed in response to a regulatory event occurring at a specific regulatory DNA sequence adjacent to or near the protein coding sequence -regulatory event involves inducer and regulatory protein -RNA polymerase initiates transcription at promoter; gene is on; protein is produced

repression

-trpR gene produces aporepressor protein (inactive repressor that alone cannot bind to the operator) -when trp is abundant in the cell, it interacts with aporepressor and converts it to an active Trp repressor -active Trp repressor binds to operator and prevents initiation of transcription -repression reduces transcription of trp operon by about 70-fold

delayed early

-uses same promoters (PR and PL) -delayed early genes are important in continuing lytic cycle -genes O and P code for proteins necessary for phage DNA replication -Q gene product, pQ, is another antiterminator, which permits transcription of late genes -genes also help to establish lysogeny -some of delayed early gene pdts are needed for integration of phage DNA into host genome -pdts of cII and cIII genes allow transcription of cI gene and production of λ repressor

regulated genes

genes whose activity is controlled in response to the needs of a cell or organism