BIOL 2301 Ch. 16

regulation of gene expression critical

major theme of molecular genetics in central dogma

all organisms cells carry same genetic info, only a subset of genes expressed in each cell type

regulation key to both unicellular flexibility and multicellular specialization

first investigated in bacteria

aspects of regulation in euk and bacteria cells similar

genes and regulatory elements

structural genes

regulatory genes

regulatory elements

in bacteria, gene regulation maintains internal flexibility, turns genes on or off in response to environmental changes

structural genes

encode proteins used is metabolism or biosynthesis

or play a structural role in cell

some constitutive

• not regulatory

• expressed continuously

regulatory genes

either RNA or proteins

interact w other DNA and affect transcription or translation of those sequences

products are DNA-binding proteins or RNA molecules that affect gene expression

used by bacteria and euks to control expression

regulatory elements

play a role in regulating genes and other DNA sequences

common in bacteria and euks

much gene regulation takes place through action of proteins produced by regulatory genes binding to elements

sequences that aren’t transcribed

regulatory genes bind to these

levels of gene regulation

regulated in a number of levels along pathway

through alteration of DNA or chromatin structure

level of transcription

mRNA processing

regulation of mRNA stability

level of translation

after translation

can be affected by regulatory activities or at these points

gene regulation through alteration of DNA or chromatin structure

only in euks

can help determine which sequences are available for transcription

rate at which sequences are transcribed

DNA methylation and changes in chromatin structure

gene regulation at transcription

limiting production of proteins early in process makes sense

transcription important point of regulation in both cell types

start transfer of info here

need to have post-transcriptional modification

for efficiency, expression often regulated early in process of

gene regulation in mRNA processing

euk DNA modified extensively before transcription

regulatory mechanisms operate at this level too

gene regulation by regulation of mRNA stability

amt of protein depends on amt of mRNA and rate at which it’s degraded

gene regulation at translation

requires large # of enzymes, protein factors, mRNA molecules

can affect rate of protein production

provide points at which gene expression can be controlled

gene regulation after translation

affect protein stability

affect whether proteins become active

regulated through processes that affect post-translational modification

DNA binding proteins

regulation in both cells accomplished by proteins that bind to DNA and affect expression

have domains

by physically attaching, affect expression

bind dynamically

never permanently attaches to DNA

other proteins compete

grouped into several types on basis of motifs

domain

discrete functional parts of proteins

consist of 60-90 amino acids

• responsible for binding to DNA

• only a few bind

some can also bind to regulatory proteins

form hydrogen bonds w DNA

motifs

simple structures

found within a binding domain

can fit into major groove

not a consensus sequence

helix-turn-helix

zinc-finger

leucine zipper

helix-turn-helix motif

2 alpha helices bound by a turn

• DNA binding and dimer binding helix connected with a turn

common in bacterial regulatory proteins

hugs minor groove

transcription factors

zinc-finger motif

euk regulatory proteins

loop of amino acids containing a zinc ion

fingers w zinc ions on them

capable of interacting w DNA

hugs major groove

transcription factors

leucine zipper motif

euk DNA binding proteins

leuicines interact as a dimer

capable of exerting action on DNA by getting between

goes between DNA helix through major groove (upside down IUD)

transcription in bacteria regulated by operons

sig diff in cell types control lies in organization of functionally related genes

bacteria genes have related functions clustered tg and under control of a single promoter

• often transcribed tg into single mRNA molecule

operon

operon

group of bacterial structural genes transcribed tg along with promoter and additional sequences that control transcription

regulates expression of structural genes by controlling transcription

chunk of DNA that contains several genes associated with single promoter

• also a set of regulatory elements

negative or positive control

inducible or repressible

lac operon

positive control and catabolite repression

trp opern

operon structure

promoter + additional sequences that control transcription (operator) + structural genes

at one end, set of structural genes

regulator gene

set of structural genes at one end of operon

A, B, and C

transcribed into single mRNA

• contents of several diff genes

• depending on bacteria, could be spliced or translated as a group

• under control of a single promoter

  • lies upstream of first structural gene

  • RNA polymerase binds then moves downstream

translated to produce enzymes, A, B, and C

• carry out series of reactions that convert precursor molecule X into product Y

regulator gene

DNA sequence-encoding products that affect operon function but not its parts

helps control expression of structural genes

• increases or decreases transcription

not considered part of operon

own promoter

transcribed into short mRNA

• translated into small protein

  • regulator protein

regulator protein

can bind to a region of the operon called operator

usually overlaps 3’ end of promoter and sometimes 5’ end of first structural gene

affect whether transcription takes place

negative and positive control

2 types of transcriptional control

operons can be inducible or repressible

negative inducible operons

negative repressible operons

positive control

negative transcriptional control

regulatory protein in the repressor

binds to DNA and inhibits transcription

positive transcriptional control

regulatory protein activator

stimulates transcription

inducible operon

transcription normally off

something must induce it

usually control proteins that carry out degradative processes

repressible operons

transcription normally on

something must repress it

usually control proteins that carry out biosynthesis of molecules needed in cell

turned off when adequate amts of product present

negative inducible operons

transcription inducible

control at operator sites negative

regulator gene encodes an active repressor that readily binds to operator

bc operator site overlaps w promoter, physically blocks binding of RNA polymerase to promoter and prevents transcription

transcription must be induced by something

inducer present and binds to repressor protein

regulates synthesis of enzymes economically

• enzymes only synthesized when substrate available

regulatory proteins have 2 binding sites

• 1 to DNA

• 1 to small molecules

inducer

binding of this alters shape of repressor

prevents it from binding

allosteric proteins

when absent, repressor binds to operator, genes not transcribed, and enzymes not produced

adaptive

turns on transcription

allosteric proteins

changes shape after binding

adaptive inducer

no precursor V present, synthesis of enzymes wasteful bc no substrate to metabolize

precursor V available, some binds to repressor and renders it inactive and unable to bind

negative repressible operons

control at operator site negative

transcription repressible

repressor synthesized in an inactive form that cannot bind to operator by itself

corepressor

inactivation of repressor allows transcription of genes and synthesis of enzymes

• results in conversion of precursor into product

repressible operon economical

• encode and synthesize only as needed

corepressor

small molecule

when present, binds to repressor and makes it capable of binding to operator

positive control

regulatory proteins involved as an activator

binds to DNA and stimulates transcription

lac operon of E. coli

first described by Jacob and Monod

• est operon as basic unit of transcriptional control in bacteria

• deduced structure of operon genetically

  • analyzed interactions of mutations that interfered with normal regulation of lactose metabolism

lactose metabolism

regulation of lac operon

lactose metabolism

lac operon regulates this

lactose metabolized by E. coli in mammalian gut

turned on in presence of lactose

lactose not easily diffused across cell membrane and must be transported by lactose permease

e. coli breaks it into glucose and galactose

• catalyzed by beta galactosidase

• thioacetamide transacetylase also produced by lac operon

  • function isn’t clear

  • could be detoxification

beta galactosidase

can also convert lactose to allolactose

• compound that plays an important role in regulating lactose metabolism

regulation of lac operon

negative inducible

b-galactosidase, permease, and transacetylase encoded by adjacent structural genes in lac operon

• common promoter

when lactose absent, few molecules of each protein produced

if lactose added and glucose absent, rate of synthesis of all 3 proteins simultaneously increases ab a thousandfold

• coordinate induction

allolactose responsible for induction

upstream of lacP theres laci which has its own promoter

laci transcribed into short mRNA translated to repressor

in absence of lactose, repressor binds to lacO

when repressor bound to operator, binding of polymerase blocked, transcription prevented

some lactose converted to allolactose when present

repression never completely shuts down transcription of lac operon

laci

own promoter

laci repressor encoding gene

lacO operon operator

• downstream from lacP

lacP operon promoter

laci transcribed into short mRNA translated to repressor

4 identical polypeptides

2 binding sites

• 1 for allolactose

• 1 to DNA

lactose converted to allolactose

binds to repressor and causes it to be released

RNA polymerase binds to promoter and moves down DNA

repressor inactivated

binding of RNA polymerase no longer blocked

transcription of lacZ, lacY, and lacA happnes

• structural genes

• lacZ encodes b-galactosidases

• lacY encodes permease

• lacA encodes transacetylase

lac proteins produced

types of lac mutations

jacob and Monod used partial-diploid strains of e. coli

structural gene mutations

regulator gene mutations

operator mutations

promoter mutations

partial diploid strains

full bacterial chromosome and an extra pace of DNA on F plasmid

• some bacteria can have extra chromosomal genes

• small circular secondary chromosome

contained lac operon

recipient bacterium became partly diploid

• possessed 2 copies of lac operon

some parts of lac operon cis acting

other parts trans acting

cis acting

able to control gene expression of genes only on same piece of DNA

trans acitng

able to control expression of genes on other DNA

structural gene mutations

some mutant strains lost ability to synthesize either b-galactosidase or permease

mutations in mutant strains mapped to structural genes and altered amino acid sequences of proteins encoded by genes

mutations affect structure of proteins but not regulation of their synthesis

• lacZ+, lacY-, /lacZ-, lacY+ produces fully functional b-galactosidase and permease

jacob and Monod able to establish mutations independent and usually affected only the product where the mutation occurred

+

wild type

/

second strand

-

not functional

regulator gene mutations

laci- leads to consitutive transcription of 3 strucutral genes

• laci+ dominant over laci- and is trans acting

• single copy of laci+ beings ab normal regions of lac operon

• laci+ lacZ- /laci- lacZ+ prodices fully functional b-galactosidase

also isolated mutation in laci gene affect prodictions of both b-galactosidase and permease

some mutations constitutive

• causes lac proteins to be produced all the time whether lactose present or not

some laci mutations isolated prevent transcription from taking place even when lactose present

• super repressors

• produced defective repressor proteins that couldn’t be activated by an inducer

super repressors

laci’s

dominant over wild type

trans acting

doesn’t bind to lactose

lacO- makes it so it can never be turned off even with super repressor

operator mutations

another class of constitutive mutations to a site adjacent to lacZ

occurs at operator

referred to as lacOc

• mutations altered sequence of DNA at operator so repressor proteins no longer available to bind

• dominant over lacO+

• cis acting

• only affects genes its physically attached to

• cannot prevent another repressor from binding to normal operators on other DNA

• laci+ lacO+Z /laci+ lacOc lacZ+ produces fully functional b-galactosidase constitutively

promoter mutations

mutations affecting lactose metabolism also been isolated at promoter

• lacP-

• interfere with binding of RNA polymerase to promoter

• don’t rpodice lac proteins regardless if alctose present or not

• cis acting

• laci+ lacP- lacZ+ /laci+ lacP+ lacZ- fails to produce fully functional b-galactosidase

positive control and catabolite repression

glucose enters glycolysis w/o further modification and requires less energy to metabolize than other sugars

when glucose available, genes that participate in metabolism of other sugars turned off through catabolite repression

catabolite repression

actually results from positive control in response to low glucose

accomplished through binding of a protein called catabolite activator protein (CAP)

CAP

site ab 22 nucleotides long

binds to promoter of lac operon and stimulates transcription

located within or slightly upstream of promoter of lac genes

RNA polymerase doesn’t bind as good w/o it

has to form a complex before binding

• cyclical amp (cAMP)

important in cellular signaling processes in both types of cells

in e. coli, concentration regulated so its inversely proportional to level of available glucose

exerts positive control on more than 20 operons

response varies

cAMP

shuts down lac operon

low when glucose high

• no positive control

high when glucose low

• positive control

binding of cAMP-CAP complex to DNA produces a sharp bend in DNA that activates transcription

In e coli, concentration of cAMP regulated so its inversely proportional to level of available glucose

higher concentration of glucose lower cAMP

RNA polymerase has poor affinity for lac promoter

little transcription of lac structural genes takes place

low concentration of glucose stimulate high cAMP

increased binding to DNA

enhances binding of RNA polymerase to lac promoter and increases transcription of lac genes approximately 50 times

trp operon of e. coli

other operons repressible

negative repressible operon

5 structural genes

controls biosynthesis of trp

when trp levels low, polymerase binds to promoter and transcribed 5 genes into single mRNA

trpR encodes repressor protein that’s normally inactive

5 structural genes of trp operon

trpE

trpD

trpC

trpB

trpA

tg they convert chorismite (og precursor) to trp

produce components of 3 enzymes

trpR

2 binding sites

• 1 for operator

• 1 for trp

binding with trp results in conformational change that makes it cpaable of binding to operator

when operator occupied, polymerase can’t bind

when trp low, transcription of operon takes place

when trp high, transcription prohibited

attenutation

transcription begins at start site

termination takes place prematurely

• attenuator sits next to anti-terminator

takes place in operons that encode enzymes participating in biosynthesis of amino acids

affects continuation of transcription, not initation

attenuation in trp operon of e. coli

best studied example in trp operon of e. coli

came to light when Yanofsky made observations that indicated repression at operator not only method of regulation

5’ UTR contains 4 regions with different complementarity in trpE mRNA

transcription when trp levels low and high differ

repression at operator not only method of regulation

isolated as series of mutant e. colis that had high levels of transcription but control at operator unaffected

some mechanism other than opression at operator controls transcription

mechanism that causes premature termination also regulated transcription in trp operon

5’ UTR contains 4 regions w diff complementariness in trpE mRNA

allow UTR to fold into 2 diff secondary structures

1 causes attenuation

• forms hairpins produced by base pairing in regions 1 and 2

• another hairpin formed with 3 and 4

• structure of bacterial intrinsic terminator includes a hairpin followed by a string of U’s

• attenuator

  • forms when trp levels high

when trp high

• 1 binds to 2

• leads to binding of 3 to 4, terminates prematurely

when trp low

• other secondary structure produced by 2 and 3 pairing

• hairpin not immediately followed by string of U’s

• doesn’t function as a terminator

• anti-terminator

  • prevents attenuator from functioning

at 5’ end upstream of region 1, ribosome binding site

• 1 encodes a small polypeptide

• 2 UGG codons that specify trp

• trp required for translation

transcription when trp levels low

RNA starts transcribing, produces region 1

ribosome binds to 5’ UTR and starts translating protein coding region

RNA polymerase transcribing region 2

2 complementary to 1, but can’t bp bc ribosome still transcribing 1

polymerase starts trasncribing 3

ribosome reaches trp codons n 1

• stalls bc trp low

• waits for tRNA with trp

• doesn’t hinder trasncription

• transcription gets ahead of translation

2 binds with 3

• forms a hairpin

• doens’t cause termination

attenuator hairpin never forms bc 3 already paired w 2

transcription continues

transcription when trp levels high

RNA polymerase starts transcribing DNA

produces 1

ribosome binds to 5’ UTR and starts translating protein coding region

when ribosome reaches 2 trp codons, keeps moving bc trp is readily available

translation keeps up with transcription

as it moves past 1, ribosome partly covers 2

polymerase finished transcribing 3

ribosome blocks pairing of 2 and 3

polymerase transcribes region 4

• pairs w 3

• produces attenuator

transcription terminated just beyond 4

no trp producing enzymes translated

no trp synthesized

key factor in controlling attenuation is # of tRNA molecules with trp bc availability determines whether ribosome stalls at trp codons

another factor is synchronizations of transcription and translation

• critical for attenuation

• achieved through a site located in 1 of 5’ UTR where polymerase pauses

• when transcribed, RNA folds into structure that prohibits transcription

ribosomes don’t transverse convoluted hairpins

why does attenuation take place in trp operon

repression never complete

some transcription initiated even when trp repressor activated

attenuation can further reduce transcription

tg, processes capable of reducing transcription more than 600 fold

attenuation and repression respond to diff signals

• repression to cell levels of trp

• attenuation to availability of tRNA w trp

trp repressor affects several operons in addition to trp operon

attenuation only refers to termination of transcription, not translation