Lecture 37: Prokaryotic Gene Regulation Flashcards
primary determinant of protein expression
DNA Transcription… aka
GENE REGULATION
basic gene unit in prokaryotes
operons
what does operon include
gene regulatory sequences (operator)
promoter
coding sequences
what do operons allow for
coordinate expression of regulated genes
lac operon regulated by
repressor protein (lac repressor) cAMP receptor protein (CRP)-- activator protein
both bind to lac operon DNA
both DNA binding proteins
trp operon regulated by
in E. coli
1) transcriptional repression
2) transcriptional attenuation mediated by ribosome pausing
why attenuate?
because transcription and translation are happening at same place and time
trp operon regulated by
in Bacillus
TRAP protein
levels of protein synth regulation
after transcription and synth of mRNA can be modulated by….
RNA turn over
rates of protein synth and turnover
long term mech of control is…
DNA transctiption (gene regulation)
if you dont need the protein product, don’t start down path of making it
prokaryote operon units
genes to be transcripbed
promoter region
1 or more regulatory sequence
allows coordinated expression of genes
transcription factors
DNA binding proteins
bind directly to DNA to regulate
binding by small molecs affects structure of proteins, which affects their DNA binding affinity
Positive and Negative Gene Regulation:
Repressors
Two ways:
1) Repressor bound to DNA w/o ligand is off=no TRX
ligand conc builds up, binds to repressor, repressor dissociates and mRNA can be transcribed
2) repressor bound to DNA W/ ligad= no transcription
ligand conc decs, ligand falls off, repressor protein dissociates, transcription happens
so what does the ligand conc do?
regulates binding events
Constititively
state the thing is in
the default state
Positive and Negative Gene Regulation:
Activators
1) protein bound to ligand, protein is ON, activates TXN, when ligand falls off, protein dissociates from DNA, txn less
2) when no ligand, protein is on, activates TXN. when ligand binds, protein dissociates, TXN less
How do represors and activators affect RNA Pol activity?
1) repressors sterically (physically) block binding of RNA pol
2) activators work molecularly to help bring things together. Protein protein interaction
Activator proteins and repressors can both…
be regulated by ligand binding
Lac operon encodes…
genes that will be involved in lactose metabolism
what is preferred carbon soruce in e. coli?
glucose
what happens when glucose is low?
we use lactose (if available) for metabolization
lac operon needed to provide the enzymes needed for lactose metabolism
lactose permease
imports lactose
beta-galactosidase
cleaves lactose into to glucose and galactose
synths low levels of allolactose (a lactose metabolite)
allolactase
inducer molecule for the lac repressor
when it binds to the repressor, it inactivates the repressor
thus turning operon on
two things needed to metabolize lactose
lactose permease
beta-galactosidase
these are part of lac operon
Parts of lac operon
promoter region
regulatory region
operator region
3 genes that encode proteins
3 genes that encode proteins
lacZ
lacY
lacA
lacZ gene
makes beta-galactosidase
lacY gene
lactose permease
lacA gene
transacetylase (not needed for lactose metabolism)
is lac operon usually on or off?
OFF (because we usually use glucose, not lactose)
but it is induceable if theres lots of lactose and no glucose
how to regulate lac operon?
binding of lac repressor
and lac operator
where does the lac repressor come from?
its around encoded by nearby DNA, but not part of operon
it is ALWAYS around, its activity depends on whether inducer molec is around
what is the inducer molec for the lac repressor
allolactase
when theres enough around, it binds to repressor, inactivates it, and turns operon on
high glucose, low lactose
operon off
lac repressor is on: binds to promoter, prevents RNA pol from binding and making lacY and lacZ genes
TRNASCRIPTION OFF
glucose and lactose high
allolactose made, binds to repressor and inactivates it (b./c lactose high)
BUT NOT TNX b/c CRP is not bound to DNA (b/c glucose high)
glucose is preferred!
TXN off (pretty much)
CRP
activator protein
needed to turn operon on
is inactive if glucose high
only active when glucose low, lactose high
where can lac repressor bind?
at multiple operator sites(3)
O1 and O2 (these play major role in repression)
lac repressor binding
tetramere
forms DNA-protein interaction
loops DNA out to prevent RNA pol from binding
when allolactose bound to repressor
HUGE conf change
this dramatically decreases DNA binding affinity
when glucose is low and lactose is high
low glucose=cAMP levels rise, bind to CRP, CRP activates DNA binding activity
high lactose= allolactose binding to repressor
active activator, inactive repressor
TRANSCRIPTION ON
active activator helps…
RNA pol bind and transcribe genes needed to metabolize lactose
what does cAMP do?
binds to CRP
turns activator on
(txn on)
binding to CRP bends the DNA
what is adenylate cyclase activated by? What does it do?
low glucose
it makes cAMP
its a bacterial thing with a glucose binding domain
Slide 11 for review
Slide 11 for review
explain why adding glucose to a bacterial culture containing lactose inhibits beta-galactose expression
because transcription is off if glucose levels are high
what happens of both glucose and lactose are low?
transcription is off
both activator and repressor are bound, but repressor wins
see experiment, almost new beta galactosidease is made
what happens of glucose is low and lactose is high?
txn is on
what happens if glucose and lactose are both high?
txn is off
regulation of E. coli trp operon
1) transcriptional repression
2) transcriptional attenuation by ribosome stalling in mRNA
how many amino acids needed to make tryptophan
5-6 depending on organism
why would E. coli use TWO mechanism to regulate trp biosynthesis?
to make sure its really really off so we don’t waste energy
there are 5 enzymes involved! It takes a lot of nrg to make these amino acids! Holy cow!
trp operon
promoter
operator
leader sequence
5 genes that make the 5 enzymes
trp repressor…
always being made
inactive in absence of tryptophan
works as repressor when there IS tryptophan
only make tryptophan if there is none
where does trp repressor bind?
at operator site
trp repressor only active repressor…
when bound to tryptophan!
when it is bound to tryp, it will bind to DNA and block transcription
leader sequence
used with the attenuation mech
part of regulatory sequence
comes before the genes for txn
4 regions
trp repressor
works just like the lac repressor pretty much
inactive when there is no tryp (so transcirption can happen)
transcriptional attenuation: 4 regions of leader sequence region 1 (lots of tryptophan)
encoders for tryptophan
when ribosome gets here, if there is enough tryp around, RNA pol just keeps synthing
transcriptional attenuation: 4 regions of leader sequence region 2 (lots of tryptophan)
if there is enough tryp around, the ribosome gets here quickly
Blocakge of sequence 2 before it can base pair with sequence 3
transcriptional attenuation: 4 regions of leader sequence
region 3 and 4 (lots of tryptophan)
if ribosome is going fast, then 3 and 4 have to base pair,
hairpin structure between them forms
what does formation of 3-4 hairpin structure do?
causes RNA Pol to fall off
so transcription of entire operon doesn’t happen
does not continue past leader sequence
we don’t make more tryptophan!
transcriptional attenuation: when tryptophan levels are low
ribosome has to wait for a charged tRNA w/ tryptophan on it
ribosome goes slowly, then stalls out while waiting, RNA pol can get a head start. ribosome doesn’t cover up sequence 2, so 2-3 can basepair
then 2-3 base pair (not really well) (doesnt make RNA pol stop)
RNA pol is not stopped, tryptophan can be made
2-3 base pair
DOES NOT act as attenuator
Regulation of Bacillus trp operon
DOES NOT USE trp repressor protein or ribosome stalling
uses large RNA binding protein
repeated sequences in region upstream of genes
what does Bacillus use of large RNA binding protein accomplish?
binds to tryptophan to halt transcription when trp levels are high
Bacillus if lots of trp
tryptophan binding to TRAP (repeated protein that looks weird, binds to repeating RNA structure)
formation of hairpin structure
txn stops
Bacillus if little trp
when transcribed to mRNA, normal structure forms, pol can read through region, txn happens
TRAP binding (bacillus)
induces RNA hairpin to terminate transcription
what might explain the evolution of yet another trp regulatory mechanism?
independent origins?
making unneeded gene products is really expensive
really need to make sure we don’t make what we don’t need
