Regulation of Gene Expression

the SteadyState Concentration of a Protein image

Gene Regulation

relies on precise protein-DNA and
protein-protein contacts

Housekeeping gene

constitutive expression

expressed in approximately all cells

Regulated gene

gene product rise and fall

inducible

repressible

Major Target of Regulation

RNA Polymerase Binding to Promoters

near the starting point of transcription initiation

RNA pol-promoter

influences the rate of transcription initiation.

Regulatory proteins enhance or inhibit this interaction between RNA pol and the promoter DNA.

E. Coli Promoters image

Substitutions in this –10 to –35 region usually reduce the
affinity of RNA Pol for the promoter.

interact with the σ factor

upstream element

interacts with the α subunit

Regulate Transcription
in Bacteria

Use of σ factors

recognize different classes of promoters

coordinated expression of different sets of genes

Binding other proteins (transcription factors) to promoters

recognize promoters of specific genes

bind small signaling molecules

posttranslational modifications

protein’s affinity toward DNA is altered by ligand binding or
posttranslational modifications

expression of specific genes in response to signals in the
environment

Most E. coli promoters recognized by σ70
.

Heat shock will replace σ70 with σ32 and direct
RNA Pol to different promoters

transcription of new products including chaperones that
keep proteins in correct conformation

Small-Molecule Effectors Can
Regulate Activators and Repressors

Repressors

reduce RNA Pol-promoter interactions

block the polymerase

bind to operator sequences on DNA

near a promoter in bacteria but further away in many eukaryotes

Effectors

bind to repressor

induce a conformational change

change may increase or decrease repressor’s affinity for the operator and thus may increase or decrease transcription

Activators

enhancers

Binding sites in DNA

In bacteria

adjacent to the promoter

bind RNA polymerase weakly

In eukaryotes

very distant
from the promoter

Regulation

Negative

involves repressors image

Positive

involves activators image

DNA Looping image

facilitated by architectural regulator proteins

Co-activators may mediate binding by binding to both activator and RNA polymerase

Many Bacterial Genes Are Transcribed
And Regulated Together in an Operon

operon is a cluster of genes sharing a promoter
and regulatory sequences

lac operon image

three genes for metabolism of lactose are
regulated together as an operon

Β-galactosidase (lacZ)

actose permease (galactoside permease; lacY)

thiogalactoside transacetylase (lacA)

They rely on negative regulation via a repressor

Lactose Metabolism in E. Coli image

glucose is abundant and lactose is lacking

Transcription is repressed

glucose is scarce and cells are fed lactose

Transcription is no longer repressed.

Inhibiting the Transcription via a Repressor Protein

a gene lacI encodes a repressor called the Lac repressor image

The repressor can bind to three operator sites (O1–O3)

binds primarily to the operator O1

prevent RNA polymerase from binding to the promoter

also binds to one of two secondary operators, with the DNA
looped between this secondary operator and O1

It reduces transcription, but transcription occurs at a low, basal rate, evenwith the repressor bound.

Induced by
Allolactose

binds to the repressor and
causes it to dissociate from the operator

[Allolactose] 上升 when[Lactose] 上升

The lac Operon Is Governed by More
Than Repressor Binding

The availability of glucose governs expression of
lactose-digesting genes via catabolite repression

mediated by cAMP and cAMP receptor protein

cAMP is made when [glucose] is low.

image

image

Combined Effects of Glucose and
Lactose on the lac Operon

When lactose is low, repressor is bound:

inhibition

When lactose is high, repressor dissociates

permitting transcription

When glucose is high, CRP is not bound and

transcription is dampened

When glucose is low, cAMP is high and CRP is bound

activation

Binding of Proteins to DNA Often Involves Hydrogen Bondingimage

Protein-DNA Binding Motifs

helix-turn-helix

zinc finger

leucine zipper

Common in DNA-Binding Proteins

one  helix for recognition for DNA , then Β
turn, then another α helix

Common in
Eukaryotic Transcription Factors

Zn2+ usually coordinated by 4 Cys, or 2 Cys, 2 His

Interact with DNA or RNA

Binding is weak so it need several zinc fingers often act in tandem

Each helix is hydrophobic on one side and hydrophilic on the other.

Approximately every seventh residue in helices is Leu (L).

Helices form a coiled coil.

The DNA-binding domain has basic residues (Lys (K), Arg (R)) to
interact with polyanionic DNA.

Eukaryotic RNA-Binding Domain

RNA recognition motifs – (RRMs)

four strand antiparallel β sheet with two αhelices

Found in gene activators and bind to both DNA and RNA

Binding to lncRNAs (long noncoding RNAs) forces the proteins
to compete with DNA for binding

decreasing gene transcription

Amino Acid Biosynthesis Regulated
by Transcriptional Attenuation

The trp operon is regulated by transcription attenuation

Transcription begins but is then halted by a stop signal
(attenuator).

in bacteria, transcription and
translation can proceed simultaneously

The attenuator sequence is in the 5’-region of a leader
sequence

can make the ribosome stall.

Role of the Attenuator

part of the leader

transcription will be attenuated at the end of the
leader

or transcription will continue into the genes for
Trp synthesis

The trp Operon image

The Leader Region Can Form
Different Stem-Loop Structures
image

If tRNATrp is abundant

segment 3 pairs with 4

attenuator

If tRNATrp is scarce

allows 2–3 pairs to form

Translation proceeds unhindered.

A Repressor Protein Also Regulates
Trp Transcription

Trp is abundant ,it binds to repressor, causes it to bind to the operator, and slows expression of genes for Trp synthesis.

It has helix-turn-helix motifs that interact
with DNA via the major groove

Regulation of the SOS Response

SOS Response image

response to extensive DNA damage

SOS genes are repressed by LexA repressor

LexA binds to operators at several genes

ssDNA is bound by the protein RecA (or, in eukaryotes Rad51).

RecA binds to LexA repressor, causing it to self-cleave and
dissociate from DNA

Synthesis of Ribosomal Proteins and
rRNA is Controlled at Translation

Ribosmal protein (r-protein) operons are regulated
via translational feedback (next slide).

Translational Feedback Mechanism

Each operon for an r-protein encodes a
translational repressor.

repressor binds to mRNA and blocks translation

Repressor has greater affinity for rRNA than for
mRNA

rRNA Synthesis Is Regulated by
Amino Acid Availability

The stringent response occurs when aa
concentrations are low.

Lack of aa produces uncharged tRNA.

Uncharged tRNA binds to ribosomal A site

rRNA synthesis triggers a cascade that begins with binding stringent factor protein (RelA) to ribosome.

Stringent factor catalyzes formation of
nucleotide guanosine tetraphosphate (ppGpp).

Binding of ppGpp to RNA polymerase reduces
rRNA synthesis.

Some RNAs Participate in Regulation

“Cis” regulation

a molecule affects its own
function

“Trans” regualtion

a molecule is affected by
another separate molecule

σS mRNA is present a low levels but is not translated due to a hairpin structure that inhibits its binding to ribosomes.

sRNAs (small RNAs) bind to this mRNA and
inhibit formation of the hairpin.

sRNA-mRNA interactions are facilitated by a
chaperone protein called Hfq.

Activation of Bacterial Translation
by Small RNA Molecules

In the presence of protein Hfq, small regulatory RNA
DsrA binds to the mRNA.

The binding of DsrA opens up the stem-loop and
allows mRNA binding to the ribosome.

DsrA RNA promotes translation.

Inhibition of Bacterial Translation
by Small RNA Molecules

In the presence of protein Hfq, small regulatory RNA
OxyS binds to the mRNA

The binding of OxyS blocks the ribosome binding site
in mRNA

OxyS RNA inhibits translation.

Cis Regulation by Riboswitches

Riboswitch = domain of an
mRNA that can bind a smallmolecule ligand

The binding of ligand affects conformation of the mRNA and its activity.

riboswitches allow mRNA to participate in their own regulation and respond to changing concentrations of the ligand.

respond to
many coenzymes, metabolites

in eukaryotic introns and
seem to regulate splicing.

target for antibiotics.

Regulation by Gene Recombination image

remove promoters relative to the coding sequence or can put genes into multiple orientations to alter the expression.

Features of Eukaryotic Gene Regulation

Access of eukaryotic promoters to RNA polymerase is hindered by chromatin structure.

requires remodeling chromatin

Positive regulation mechanisms

Three Features of Transcriptionally Active Chromatin

nucleosomes repositioned

histone variants

covalent modifications to nucleosomes

Nucleosomes Can Be Restructured by
Specific Protein Complexes

SWI/SNF (SWItch/Sucrose NonFermentable)
complex

remodels chromatin

stimulates binding of transcription factors

works with proteins of ISWI (imitation switch)
family

ATP-dependent alteration of spacing between
nucleosomes

Histone Modification Alters Transcription

Methylation of Lys-4 and Lys-36 at histone3 (H3) and
Arg of H3 and H4:

recruits histone acetyltransferases (HATs) that then
acetylate a particular Lys

reversed by histone deacetylases (HDACs) that make
chromatin inacti

Acetylation of Lys results in decreased affinity of histone
for DNA.