Basic Transcription Part 2 Flashcards
operon
- group of contiguous, coordinately controlled genes
inducible gene expression
- certain genes expressed under certain environmental conditions
cells will go on which first
- glucose
- then lactose use lac operon
one strategy to regulate gene expression
- group functionally related genes together
Beta-galactosidase
- used to break down lactose into glucose and galactose
all 3 genes transcribed together from
- 1 promoter to produce
- 1 mRNA = polycistronic mesage
each gene has its own
- ribosome binding site
lacY
- permease
- transports lactose into the cell
LacA
- transacetylase
lac operaton under negative control
- by the lac I repressor
lac I repressor
- tetramer of 4 identical polypeptides
- binds to operator next to promoter
- binds to operator and prevents RNA polymerase from binding to promoter and transcribing
repressor is an allosteric protein
- binding of one molecule changes shape of remote site
- allows interaction with a 2nd molecule
inducer
- binds repressor
- causes change in confirmation and dissociation from the operon.
- is lactose or allolactose
discovery of lac operon
- certain cryptic mutants expressed B-galactosidase, but could not metabolize lactose
- add radioactive galactoside to wild type and mutant bacteria
- only induced wild type took it up
jacob and monod suggest
- substance induced with b-gal to get galactosidase into cells
- mutants defective in this gene
- that gene was permease
constitutive mutants
- produced all 3 gene products all the time without induction
- also not capable of being repressed
idea of a repressor
- created merodiploids
merodiploids
- partial diploid bacteria
- had both wild-type (inducible) and constitute alleles
- wild-type dominant
- could turn off genes from constitutive and inducible parent
- wild type produces something that keeps lac genes off unless they are induced
predicted the existence of 2 key control elements
- repressor
- operator
deletion mutants
- also revealed promoter needed for expression of all 3 lac genes
existence of a repressor suggested
- a DNA sequence that binds the repressor
- operator
4 types of mutations
- (I^-) - recessive
- (O^c) - cis-dominant
- (I^s) - dominant in cis and trans
- (I^d) - uninducible
recessive mutation in the repression (I^-)
- I- mutations make a repressor that cannot recognize the operator
- mutations that eliminate the function of the repressor are recessive
- if combined with wild type will make inducible and repressible again
operator constitutive mutation (O^c)
- mutations in 1 of operators cause it to be active (unresponsive to repressor)
- other operator still repressible
- cis-dominant mutation
I^s mutations
- mutation in lac repressor
- some subunits can bind allolactose some cannot (make unresponsive to inducer)
- will poison system so neither lac operon will be inducible
- dominant in both cis and trans
I^d
- defect product can form defective tetramers with wild type repressor
- won’t bind operator
- no way to repress operon
- whole tetramer inactive
- dominant negative (dominant in cis and trans)
repressor-operator binding
- add more repressor in presence of inducer - poor binding
- add more repressor in absence of inducer get increased binding till it levels off
- repressor binds to operator nicely in absence of inducer
O^c lac operator binding to repressor
- binds with lower affinity
- O^c lac operator requires more repressor for binding
what activates lac operon
- activation needed only in absence of glucose
glucose present
- repression keeps lac operon off
as glucose level drops
- cAMP level increases
Catabolite activation
- cAMP can activate lac operon even when some glucose is present
catabolite activator protein (CAP)
- binds to cyclic AMP
map near lac promoter
- at an activator site
- just upstream of promoter instead of downstream like activator
- called CAP binding site
- alpha-CTD of pol binds
cAMP and CAP effect on DNA
- cause the DNA to bend
- stabilizes the interaction
DNA bending leads to
- induced fit
