Lac Gene Regulation Flashcards
why do bacteria regulate gene expression?
bacteria have to change their physiology to adapt to changes in their environment to maximize growth and preserves energy and resources.
what is the effect that adaptation has on the bacteria?
there are structural and functional changes that require different proteins
what are the two main ways that gene expression is regulated?
i) changing RNA levels (leads to changes in protein levels)
ii) on/off switching of gene (induction/repression)
generally (really briefly) how does transcriptional regulation occur in prokaryotic cells?
Gene is generally on (activator protein encourages RNA pol binding to promoter). Gene is off when repressor protein binds to operator/promoter preventing RNA poly from binding
generally (really briefly) how does transcriptional regulation occur in eukaryotic cells?
Gene is generally off with DNA wrapped around histones preventing RNA pol binding. Gene is turned on when transcription factors bind to enhancer sequences allowing RNA pol to bind to TATA box and proceed with transcription
What are the different levels of regulation in bacterial gene expression? + ways that it occurs?
-transcriptional control (change rate of transcription)
-translational control (change translation rate & mRNA stability)
-post-translational control (protein modification, stability, location)
when does most gene expression regulation in bacteria occurs? why?
-transcription initiation
-this is the most efficient point of regulation because of the cell doesn’t waste resources/time starting a process that it doesn’t need at the moment
according to the central dogma, what are some of the ways that regulation can occur at the transcription level?
-alternative sigma factors
-repressors
-activators
according to the central dogma, what are some of the ways that regulation can occur at the translation level?
-small antisense RNAs
-RNA 2 structure
-RNA stability/half-life
according to the central dogma, what are some of the ways that regulation can occur at the protein level?
-protein stability/half-life
-localization/secretion
-modification
what is negative gene regulation?
decrease gene expression
what is positive gene regulation?
increase gene expression
where does the repressor protein bind in the gene and what activity does it do? what type of regulation is the repressor protein involved with?
-operator/overlaps with promoter (steric hinderance prevents RNA pol binding)
-turns off gene expression
-negative regulation
where does the activator protein bind in the gene and what activity does it do? what type of regulation is the repressor protein involved with?
-activator site/upstream -35
-turn on gene expression
-positive regulation
what is the general structure of gene?
-activator site (-35)
-promoter (has operator at the end)
-genes (can produce polycistronic mRNA)
what is constitutive gene expression?
genes are expressed continuously
Inactivation mutation of repressor results in what type of gene expression? Mutation that inactivates repressor is ____ (gene interaction) to WT. Why?
-constitutive gene expression (repressor can’t bind & activate gene expression)
-recessive
-presence of WT version of gene in plasmid can produce WT phenotype cause WT repressor produced can still act on gene to repress transcription
Inactivation of activator results in what type of gene expression? Mutation that inactivates repressor is ____ (gene interaction) to WT. Why?
-non-inducible gene expression (activator can’t bind/activate gene expression)
-recessive
-presence of WT version of gene in plasmid can produce WT phenotype cause WT activator produced can still act on gene to activate transcription
Gain-of function mutation of repressor results in what type of gene expression? Mutation that make repressor always activate is ____ (gene interaction) to WT. Why?
-non-inducible gene expression (repressor always bind/active so gene can’t be induced)
-dominant
-repressor is always active so it also represses WT version of gene in plasmid
Gain-of function mutation of activator results in what type of gene expression? Mutation that make activator always activate is ____ (gene interaction) to WT. Why?
-constitutive gene expression (activator always binds/activates gene expression)
-dominant
-activator is always active so it also activates WT version of gene in plasmid
Who discovered Lac gene regulation winning the 1965 Nobel Prize in Medicine & said that “Anything found to be true of E.coli must also be true of elephants”?
Francois Jacob & Jacques Monod
Generally gene review of Lac Operon?
Lac I (produces repressor)
(promoter + operator)
LacZ (B-galactosidase)
LacY (Permease)
LacA (Transacetylase) - uptake of galactose
what are the products of regulatory genes? What do the products of regulatory genes do?
-repressors/activators
-control the transcription of genes/operons
how do repressors/activators regulate transcription?
bind DNA close to promoter using conserved Helix-turn-helix DNA binding motif
How did Jacob & Monod characterized the genes responsible for degradation of sugar lactose?
1) they isolated hundreds of lac- mutants
2) they constructed merodiploids (partially diploid bacterium that has chromosome & chromosome fragments)
3) they performed complementation analysis
how did Jacob & Monod isolate Lac- mutants?
1) They used color indictor plates -> macconkey lactose plates have pH sensitive dyes that turn colour if sugar is metabolized so white colonies are Lac- and pink colonies are Lac+
2) X-gal is a colourless B-galactoside that turns blue when cleaved by B-galactosidase, a product of lacZ gene
How did Jacob & Monod construct merodiploids?
they transferred some mutations to a F’ lac plasmid by recombination & made partial diploids by conjugation
How to read this genotype statement: F’lac-/lac+?
F’ lac- = plasmid genotype (lac mutant)
lac + = cell genotype (lac WT)
How does complementation analysis work?
F’ plasmid has lac mutation; Chromosome also has lac mutation. Merodiploids constructed with F’ plasmid will have two of different phenotypes.
Lac + = the two recessive mutations complement each other so the mutations must be in different genes. (gene products on one molecule cover the mutation in other DNA molecule)
Lac - = the two recessive mutation don’t complement each other so the mutations are in the same gene
What does complementation analysis generally determine?
the number of gene products involved (# of complementation groups)
dominance vs recessiveness (aka the function of gene products)
identity of cis vs trans factors (sites vs. proteins)
In complementation analysis, if the phenotype of the mutant allele is observed, what is the nature of mutant allele? what if phenotype of wild-type allele is observed?
-dominant
-recessive
In Jacob & Monod analysis, the majority of Lac- mutants were ____ (gene expression) with WT lac genes
-recessive
What were the two genes products identified with complementation analysis that were necessary for lactose metabolism?
LacZ -> B-galactosidase
LacY -> lactose permease
Are the lac genes inducible or constitutively active? What observation lead to this conclusion?
-inducible
-absence of lactose -> low levels of lacZ & lac Y mRNA
-presence of lactose (inducer) -> B-galactosidase & lactose permease synthesized simultaneously
lacI- mutants are constitutive and ______ to WT because WT LacI gene likely encodes what kind of product?
-recessive
-diffusible product that can repress both lac operons in merodiploid
-it encodes diffusible product, likely a protein molecule that represses lac operons on both DNA molecules
LacI encodes what kind of protein molecule that works in _____.
-repressor
-trans
lacOc mutants are constitutive and ______ to WT but dominance is only shown in certain combinations of lac mutations.
-dominant
LacOc mutants display cis dominance. What does that mean?
-LacOc only affects expression of lac genes of same DNA molecule meaning that the gene does not encode diffusible product.
-ex. mutant LacOc on plasmid won’t affect WT LacO on chromosome
What is the Lac O gene?
LacO is a regulatory site on DNA that controls lacZ and lacY expression; cannot be complemented; lacI repressor binds to lacO
what is the mapping of lac operon?
-lac I
-lac P (promoter)
-lac O (operator)
-lac Z
-lac Y
What is the Operon Model proposed by Jacob & Monod to explain regulation of lac genes?
1) lactose utilization need lac operon which has structural genes (lacZ & lacY) for lactose uptake & breakdown and regulatory elements that control inducible expression
2) lacI gene makes repressor that binds to lacO operator
3) without the inducer lactose, LacI binds to operator and prevents RNA pol from transcribing lacZ & lacY
4) with lactose, lacI is inactivated by lactose binding so it can’t bind to operator, and lacZ and lacY are transcribed by RNA poly
`what are the non-inducible mutants of lac operon? (3)
-lacZ-, lacY-
-lacIs = lac I super-repressor
-lacP- = promoter mutants
lacIs mutants make no lac mRNA, even in presence of the inducer. F’ lacIs/lacI+ = non inducible -> lacIs is ___ to WT. What is the current model of lacIs mutant?
-dominant
-lacIs makes a mutant repressor that either fails to bind inducer or that binds inducer but fails to dissociate from operator
New lac gene developments: third gene in operon
lac A gene encodes a transacetylase not required for lactose utilzation
New lac gene developments: true inducer
allolactose (metabolite of lactose)
New lac gene developments: lacI repressor can bind to 3 different operators
O1, O2, O3 but O2 and O3 are not necessary but they might enhance repression through loop formation
New lac gene developments: lacI repressor structure
crystal structure of lacI has been solved
positive regulation of lac operon was identified by mutations in what genes? what are the phenotype of mutations in these genes?
-crp and cya genes (these genes are distant from lac operon)
-crp(-) = lac-, non-inducible
-cya(-)= lac-, non-inducible
-F’crp-/crp + = lac+, inducible
-F’ cya-/cya+ = lac +, inducible
Crp & Cya mutants have opposite phenotype to lacI- mutations which suggests what?
the products of crp & cya genes are required to activate expression of lac genes
what is the gene product of Cya? what does it do?
-adenyl cyclase
-synthesize cAMP when [glucose] = low
what is the gene product of Crp? what does it do?
-activator protein
-cAMP binds to it wheb glucose is low to promote transcription of lac operon
CRP stands for what? aka what?
-cAMP Receptor Protein
-Catabolite activator protein (CAP)
How does regulation by CRP/CAP & Cya works?
1) Cya makes cAMP when [glucose] = low
2) cAMP binds to CRP
3) CRP: cAMP binds region upstream lac operon that interacts with RNAP to activate transcription of lac operon
4) this ensure genes for lactose metabolism are not expressed if glucose (the preferred carbon source) is present
Lac gene expression requires two things?
low glucose and presence of lactose
CRP + cAMP are known as a global regulator because of what?
it regulates the expression of other operons whose products are involved in breakdown of other sugars
