Genet 270 - gene reg - topic 1 lac operon

why is gene expression regulation important for bacteria?

• adaptation (to environmental changes)

• preserves energy

• provides maximum growth

• produces structural + functional changes (requires different proteins)

what is gene expression regulation accomplished by?

• RNA levels

• on/off switching (induction/repression)

how many levels can gene expression be regulated at? what are they?

• 3 levels

• transcriptional control, translational control, and post-translational control

what is the most efficient point of regulation?

transcription initiation

the repressor regulatory protein is what type of regulation?

negative

the activator regulatory protein is what type of regulation?

positive

how do repressors regulate gene expression?

binds operator overlapping promoter which blocks RNAP → turning gene expression OFF

how do activators regulate gene expression?

binds activator site upstream (-35 region) from promoter which recruits RNAP → turning gene expression ON

what conserved DNA binding motif is used by repressors and activators?

the helix turn helix DNA binding motif

Genetic technique for distinguishing the negative regulation of inactivation

constitutive expression, recessive

Genetic technique for distinguishing the positive regulation of inactivation

non inducible expression, recessive

Genetic technique for distinguishing the positive regulation of gain of function

constitutive expression, domininant

Genetic technique for distinguishing the negative regulation of gain of function

non inducible expression, dominant

what do repressors and activators do?

bind DNA close to promoter to regulate transcription, used conserved helix turn helix DNA binding motif

what did jacob and monods aproach do?

characterize genes responsible for degradation of lactose

how did jacob and monod characterize the genes for the degradation lactose

isolated Lac- mutants

constructed merodiploids

preformed complementation analysis to determine number of gene products involved dominants vs recessiveness and identity of cis vs trans factors

what was the results of jacob and monods aproach?

id of structural genes lacZ and lacY

majority of Lac- were mutants recessive in merodiploid with wild type lac genes

what does lacZ incode?

beta galactosidase

what does lacY incode?

lactose permease

are levels of lacZ and lacY MRNA low or high when there is an absence of lactose?

low

What happens in the presence of lactose

beta galactosidase and lactose permease synthesize simultaneously (lactose is inducer)

what are the 2 constitutive mutants?

lacI and lacOc

whats the phenotype of lacI- mutants

constitutive recessive

what does lacI encode and does it work in cis or trans

encodes a repressor and works in trans

what does the phenotype for lacOc mutants

constitutive dominant (dominance is evident only in certain combinations of lac mutations)

what is the function of the diffusible product of lacI gene

it is a repressor that binds to the operator (lacO in the absence of the inducer preventing RNAP from initiating transcription)

what is the true inducer molecule that binds to and inactivates the LacI repressor

allolactos, a metabolite of lactose

what does constitutive mean?

always on

what does cis- dominance mean

the mutation only affects genes on the same DNA molecule and defines a regulatory site

describe the phenotype and key genetic characteristic of a lacIs ( super repressor) mutant

Non inducible and dominant (this repressor either fails to bind the inducer or fails to dissociate from the operator even when the inducer is present)

what is the modern view of the lac operon

the paradigm for gene regulation

what are the new lac developments

1. third gene in operon = lacA (encodes transacetylase not required for lactose utilization)

2. true inducer = allolactose

3. lacI repressor can bind to 3 different operators (O1 O2 O3)

   1. crystal structure of LacI has been solved

what is the overall purpose of catabolite repression in regulating the lac operon

insures lac genes are not expressed if a better carbon source ( glucose) is available (glucose is the preferred source)

what protein acts as the activator in positive control and what is its effector molecule

CRP (cAMP receptor protein) also known as CAP its effector molecule is cAMP

when are cAMP levels high enough to activate lac gene transcription

only when glucose levels are low because Cya synthesizes cAMP only under this condition

what 2 specific conditions must be met for high level lac gene expression

low glucose and presence of lactose

what is the function of Crp

its an activator protein (cAMP receptor protein)

what is the function of cAMP

crp activator effector molecule

how does crp activate transcription

camp binds region upstream of lac operon then interacts with RNAP which activates transcription

what is the function of catabolite activator protein ( CAP)

insure genes for lactose metabolism not expressed if a better carbon source is available

what is the global regulator

crp + cAMP