Genetics: Bacterial Gene Control

Genetics: Bacterial Gene Control

constitutive gene expression

genes that are always active, their product is in constant demand

regulated gene expression

- their products are rarely needed
- only expressed when needed
genes are hardly ever active

operons

genes that coordinate the regulation of gene expression

polycistronic and operons

several genes transcribed from same promoter and regulated by the same conditions
make products that function in similar ways

structural genes

- genes that are to be transcribed
- may be enzymes or structural proteins
- these genes are occasionally needed

Promoter (P)

- site that is a fixed # nts upstream
- -35, TATA, etc boxes located here
- place RNA polymerase recognizes
- remember: all genes have a promoter-- in operon or not

Operator Site (O)

- site that is fixed in place, near or adjacent to the initiation site of operon structural genes
- site which determines if transcription will occur
- may be more than 1 present

what a protein regulator will bind to

Regulator (R)

- protein that acts at the operator site to regulate transcription
- gene encoding regulator does NOT have to be near operon genes
- regulators may be represented with other letters/ info

Operon organization

1. inducible
2. repressible

both of these can have positive and neg regulation
for repressible mostly neg

positive regulator recruits RNA polymerase by:

1. direct interaction
2. making promoter accessible

Negative regulation blocks RNA polymerase by:

- positive and negative regulation describe effect of regulator on transcription
- inducible system can have both types of regulation

inducer molecules cause

transcription in both regulation types

positive regulation on gene x without inducer

activator cannot bind DNA --> no transcription

positive regulation on gene x with inducer

activator and inducer bind and are able to bind to DNA which allows for transcription to happen --> mRNA

negative regulation on gene Z without inducer molecule

the repressor remains on gene Z and no transcription occurs

negative regulation on gene Z with inducer molecule

inducer molecule binds the repressor so it cannot bind to DNA
Gene Z continues on with transcription and mRNA is made

inducible systems: keyed to substrates (Gene X)

Gene X - often encodes catabolic enzyme
Substrate serves as inducer of gene X expression

Repressible systems: keyed to end products (Gene Z)

Gene Z - often encodes anabolic enzyme
End product of enzyme serves as a repressor of gene Z expression

lac operon

- first operon to be discovered
- inducible
- contains 3 structural genes > each encode for an enzyme that allows the cell to use lactose as an energy source

lac operon expression

genes are expressed unless repressor is bound at the operator

if E. Coli are grown in the presence of glucose

then the glucose is suddenly replaced with lactose

E.coli in which glucose is replaced with lactose

- cells will seem unable to metabolize lactose for short time
- lag in growth, cells appear unable to survive
- during this time the cells are turning on the lac operon

3 critical genes of lac operon

Z- ß-galactosidase
Y- permease
A- transacetylase

Z- ß-galactosidase

breaks lactose into 2 monosaccharides (glucose and galactose)

Y- permease

transports lactose into cells

A- Transacetylase

transfers an acetyl group from acetyl-CoA to galactosides; prevents buildup of toxic product of ß-galactosidase

LacI+ gene

encodes the operon regulator protein

Protein made from lacI+

bound to operator unless inducer molecule is present (lactose)

what kind of regulation is lac operon under

positive and negative

negative regulation of lac operon

implies something is turning off gene transcription by binding at a control element

what is the repressor of lac operon

protein encoded by regulatory site lacI

in the absence of lactose the Lac operon is

mostly off
repressor is bound at the operator when lactose is not present

lac operon expression on vs off

on: lactose present and glucose is not
off: glucose is present

homotetramer repressor

- can bind 2 of 3 operator sequences at once (O1, O2, O3)
- if bound to O1 and O3, intervening DNA forms a repressor loop

maximum repression of lac operon occurs when

all 3 O sites are bound
- lacO2 is at +412

which gene is expressed at low levels constitutively in lac operon

repressor gene
- has weak promoter
- a few repressors always bound
- repressor molecules bind and unbind
- even without inducer present, RNA pol. could initiate transcription before another repressor binds

what must happen to repressors for operator to turn an operon on

they must be prevented from binding operator

lac operon in presence of lactose

allolactose binds the repressor

allolactose acts as what

the inducer molecule

allolactose binds the repressors:

- some lactose is converted by ß-galactosidase into allolactose
- binds repressor at operator, changes its shape
- also binds free repressors

what is more efficient for the cell to use? lactose or glucose?

Glucose

Energy for biochemical reactions comes from

Glucose

operon is at basal level unless

something intervenes to turn it on
- neg control is like a brake
- removing brake is not enough to activate expression

How does E. Coli keep lac operon at basal level if glucose is present

- influence of a breakdown product, a catabolite of glucose metabolism
- called catabolite repression or glucose effect

what is the best positive controller of the lac operon

a substance that senses the lack of glucose and activates the operon

lac operon: catabolite repression with cAMP

- cAMP formed from cytosolic ATP by adenyl cyclase
- cAMP has numerous roles in signal transduction pathways
- Glucose inhibits adenyl cyclase
- cAMP has a crucial role in catabolite repression
-↑ [glucose] results in ↓ [cAMP]= basal lac operon transcription

what happens if cAMP is added to bacteria

it can overcome catabolite repression of the lac operon (even in the presence of glucose)

cAMP also has a role in positive control of

gal and ara

process of positive control is complex, what are the two parts?

1. cAMP
2. protein factor

CAP

catabolite activator protein
also called cAMP receptor protein

gene name of CAP

crp

Mechanism of CAP and cAMP action

- When present in high amounts, CAP and cAMP associate
- This complex binds just 5 ́ of operon promoter
- Now RNA pol can strongly recognize promoter and considerable transcription takes place

The CAP binding sites in lac, gal, and ara operon promoters all contain what sequence

TGTGA

What sequence is important for the CAP-cAMP complex binding

TGTGA

Operons activated by CAP and cAMP have remarkably weak promoters

- their -35 boxes are not very similar to consensus sequences
- almost not recognizable

Why does weak promoter at lac operon make sense

> constitutively, basal expression level
- glucose levels drop, cAMP rises
> cAMP associates with CAP, complex
- makes stable RNA polymerase promoter complex
- increased production of lactose digesting enzymes

what does it mean that the lac operon is still under operator control

if repressor binds, basal level occurs

FINAL regulation of lac operon depends on what

presence of inducer:
appropriate inducer specifically selects which operon will work

lac operon: cAMP present and lactose present

CAP and cAMP are bound together, lactose makes the repressor inactive
>> binding of RNA pol is facilitated by CAP and transcription occurs

lac operon: cAMP present and lactose absent

CAP is bound to cAMP but without lactose the repressor is active and binds >> transcription is blocked by represor

lac operon: cAMP is absent and lactose is present

CAP is inactive and repressor is inactive >> transcription inhibited by lack of CAP

lac operon: cAMP is absent and lactose is absent

CAP is inactive and the repressor is active >> transcription inhibited by lack of CAP and the presence of repressor

lac operon mRNA translation

- structural genes in lac operon close together
- once ribosome is loaded onto transcript, it does not release until all 3 genes are translated
- once ribosome reaches UGA of one gene, it stays on long enough to reach AUG of next gene

nonsense mutation in structural gene

- ribosome will not reach next AUG
- genes following nonsense mut. will not be translated

making stop codon mutation where one shouldn't be

polar mutations (polar effects)

always only affect downstream

missense mutations

point mutation, changes one amino acid into another amino acid

Conjugation: the F-plasmid

Physical contact between 2 cells is essential*
• Contact is the initial step
- established by structure called F sex pilus
- F+ cells have cell extensions covering external surface = F-pili
- Once a pilus is in contact with F- cell, it elongates and becomes an F sex pilus

Conjugation of the F plasmid

a copy of the F factor is almost always transferred from F+ to F- cell, converting it to F+ state

F' Factor (lac)

plasmid formed of the F factor + bacterial gene that loop out of the chromosome

transfer of an F' factor during conjugation

carries some of the same genes already in the recipient's chromosome

merozygote or partial diploid

a partial diploid strain of bacteria containing F' factor genes

mutations that change the operator sequence so repressors cannot bind

- results in constitutive expression of the structural genes
- called operator-constitutive mutations (lacO^c)
- if partial diploid has a lacO^c and lacO+
>> only lacO^c will cause constitutive expression of struct genes
>> other copy expressed normally
>> called cis-dominant mutation

LacO^c changes the operator sequence so

repressor cannot bind

LacI- changes the regulator sequence so

repressor cannot bind operator
also causes constitutive operon expression

partial diploid with lacI and lacI-

- WT regulator gene makes WT repressor
- WT repressor can bind both copies of operator
- lac+ regulator sequence can overcome lacI- mutation
- lacI+ gene has trans-acting function to lacI- gene

superrepressor lacI^s changes the regulator sequence so

regulator can bind operator but not allolactose

once the lac^s repressor binds to operator

it cannot be induced to fall off
- results in basal transcription level even in presence of lactose

Superrepressor is trans-acting protein which means that

LacI^S dominant to LacI+

lacP changes the promoter so

RNA polymerase cannot bind

- -10 or -35 box mutation
- cis-acting function

LacI^d changes the regulator sequence so

subunits cannot form complete repressor
- repressor has homotetrameric structure

in partial diplods that are lac^-d/ lacI+

- if 1 or more subunits are lac^-d then, repressor will not form native confirmation
- repressor function will be lost
- ~12 repressor molecules in cell, so 1 mutant subunit blocks normal operator binding
- lacI^-d subunits trans-dominant to lacI+ subunit

trp operon

- contains the genes that code for enzymes that E. coli needs to make amino acid tryptophan
- negatively controlled by a repressor

Fundamental difference between lac and trp operons

- lac operon codes for catabolic enzyme (break down substance)
- such operons are turned ON by the substance (like lactose)

- trp operon codes operon codes for anabolic enzymes (builds up a substance)
- such operons are turned OFF by substance made (like tryptophan)

trp operon: genes

operon codes for 5 polypeptides that form 3 functional enzymes
- termed gene trpE, D, C, B, A

Trp E and trp D

code for polypeptides that make first enzyme

trp C

codes for single polypeptide enzyme that catalyzes intermediate steps

trp B and trp A

code for 2 polypeptides of enzyme that catalyzes last 2 steps

where does the trp operon lie

within the promoter region

the lac operon operator was where?

adjacent to the promoter

the presence of Trp means

the operon needs to shut down
- Trp acts like a corepressor

corepressor

it is half the functional repressor molecule

aporepressor

Trp can bind to a protein

aporepressors need whag\t

Trp to function

aporepressor + corepressor

binds operator and shuts off operon

low Trp concentration

inactive repressor allows transcription

high Trp concentration

active repressor complex binds to operator and prevents transcription

Trp bound to aporepressor forms active complex

causes conformational change in aporepressor to allow DNA binding

trp operon is

repressable
very common in prokaryotic a.a. synthesis pathway

what is the problem with this type of repression (repressable)?

- repressor/operator association is weak
- even at high [Trp], operon is still working at appreciable level

A second regulation mechanism:

ATTENUATION