Download RNA polymerase I

Chapter 15.
Regulation of Gene
Expression
RNA Polymerase is the Key Enzyme for
Transcription
• RNA polymerase involved in three phases of transcription.
1) Initiation : binds to promoter, unwinds DNA, begins
polymerization of bases complementary to DNA template.
2) Elongation : movement away from promoter sigma subunit
released, polymerization.
3) Termination : signal reached by RNA polymerase.
- Rho dependent termination : Rho factor recognizes
sequence in mRNA, binds to it, and pulls it away from RNA
polymerase.
- Rho independent termination : stem loop structure formed
by sequence of 20 bases with a run of 6 or more U’s signals
release of RNA polymerase.
The Regulation of Gene Expression Can
Occur at Many Steps
• Binding of RNA polymerase to promoter
• Shift from initiation to elongation
• Release of mRNA at termination
• Posttranscriptional stability of mRNA
• Efficiency of ribosomes to recognize translation initiation sites
• Stability of polypeptide product
The Utilization of Lactose by E. coli: A
Model System for Gene Regulation
• The presence of lactose induces expression of the genes
required for lactose utilization.
– Induction : stimulation of protein synthesis
– Inducer : molecule that stimulates synthesis
– Lactose : inducer of genes for lactose utilization
• 1950s and 1960s – Golden era of bacterial genetics
• Advantages of E. coli and lactose utilization system :
1) Culture large numbers of bacteria allow isolation of rare
mutants
2) Lactose genes not essential for survival (can use glucose as
carbon source)
3) Induction increases expression 1000 fold making mutant
identification easy
Coordinate Repression and Induction of
Three Genes Revealed by Studies of LactoseUtilization Mutants
• Jacques Monod and Francois Jacob (Pasteur Institute in
Paris) : Proposed ‘Operon Theory’ of gene regulation.
1) Single signal can simultaneously regulate expression
of several genes that are clustered together on a
chromosome and involve the same process.
2) Because genes are clustered, they are transcribed
together as single mRNA.
3) Clusters of genes are called operons.
Complementation Analysis of Mutants
Identifies Lactose Utilization Genes
• Monod et al. isolated many lac- mutants unable to
utilize lactose.
• Complementation analysis identified three genes (lacZ,
lacY, and lacA) in a tightly linked cluster.
• In 1961, Francois Jacob and Jacques Monod proposed
the operon model for the control of gene expression
in bacteria.
• An operon consists of three elements:
– the genes that it controls,
• In bacteria, the genes coding for the enzymes of
a particular pathway are clustered together and
transcribed (or not) as one long mRNA molecule.
– a promoter region where RNA polymerase first binds,
– an operator region between the promoter and the
first gene which acts as an “on-off switch”.
Experimental Evidence for Repressor
Protein
• Isolated mutant in lacI gene
– Constitutive mutant : synthesized βgalactosidase and lac permease even in absence
of lactose (inducer).
– lacI must be a repressor : cells must need lacI
protein product to prevent expression of lacY
and lacZ in absence of inducer.
Inducer Releases Repressor to Trigger
Enzyme Synthesis
• Addition of lactose inducer caused β–galactosidase
synthesis to continue.
• Conclusion: Inducer binds to repressor so repressor
can not bind to DNA.
• Allosteric effect : inducer bound to promoter changes
conformation of protein so it can not bind to DNA.
Repressor has Binding Domains for
Operator and for the Inducer
Changes in the Operator can also
Affect Repressor Activity
Proteins Act in trans
DNA Sites Act Only in cis
• Trans acting elements : can diffuse through
cytoplasm and act at target DNA sites on any DNA
molecule in cell.
• Cis acting elements : can only influence expression
of adjacent genes on same DNA molecule.
Regulatory Elements that Map Near a
Gene are cis-acting DNA Sequences
• cis-acting elements
– Promoter : very close to gene’s initiation site
– Enhancer :
• can lie far way from gene
• can be reversed
• augment or repress basel levels of transcription
lac Operator
cis-elements of lac Operon
Regulatory Elements that Map Far from a
Gene are trans-acting DNA Sequences
• Genes that encode
proteins that interact
directly or indirectly with
target genes cis-acting
elements.
– Known genetically as
transcription factors
– Identified by :
• Mapping
• Biochemical studies
to identify proteins
that bind in vitro to
cis-acting elements
Repressor vs. Activator
Activator
Model showing how the lac repressor (red) and catabolite activating
protein (dark blue) bind to the lac operon promoter, creating a 93-basepair repression loop in the lac regulatory DNA.
• Repressible enzymes generally function in anabolic
pathways, synthesizing end products.
– When the end product is present in sufficient
quantities, the cell can allocate its resources to
other uses.
• Inducible enzymes usually function in catabolic
pathways, digesting nutrients to simpler molecules.
– By producing the appropriate enzymes only when
the nutrient is available, the cell avoids making
proteins that have nothing to do.
• Both repressible and inducible operons demonstrate
negative control because active repressors can only
have negative effects on transcription.
The Operon Theory
• The players:
– lacZ, lacY, lacA genes that split lactose into glucose and galactose
– Promotor site to which RNA polymerase binds
– cis acting operator site
– trans-acting repressor that can bind to operator (encoded by lacI
gene)
– Inducer that prevents repressor from binding to operator
A simplified overview of the genes and regulatory units involved in the
control of lactose metabolism. (This region of DNA is not drawn to
scale.)
The components of the wild-type lac operon
The gratuitous inducer isopropylthiogalactoside (IPTG).
The structural genes of the lac operon are transcribed into a single
polycistronic mRNA, which is translated simultaneously by several
ribosomes into the three enzymes encoded by the operon.
The catabolic conversion of the disaccharide lactose into its
monosaccharide units, galactose and glucose.
Repression
• Repression
– In absence of lactose, repressor binds to operator which
prevents transcription.
– Negative regulatory element
The response of the lac operon to the absence of lactose.
Induction
• Induction
– Lactose present
• Allolactose binds to
repressor.
• Repressor changes
shape and can not
bind to operator.
• RNA polymerase binds
to promotor and
initiates transcription
of polycistronic mRNA.
The response of the lac operon to the presence of lactose.
Inducible synthesis
•
lacI+ gene encodes a diffusible element that acts in trans by binding to any
operator it encounters regardless of chromosomal location.
Molecular Studies Help Fill in Details of
Control Mechanisms
• Radioactive tag attached to
lac repressor.
– Repressor from lacI+ cells
purified and mixed with
operator DNA, cosediment occurred.
– Repressor from lacI+
mixed with mutant
operator DNA, no cosediment occurred.
The response of the lac operon in the absence of lactose when a cell
bears the OC mutation.
Constitutive
•
•
Presence of O+ plasmid does not compensate for Oc mutation on bacterial
chromosome.
Operator is cis acting element.
The response of the lac operon in the absence of lactose when a cell
bears the I- mutation.
The response of the lac operon in the presence of lactose in a cell
bearing the IS mutation.
Noninducible
•
•
•
All operator sites (O+) eventually occupied by superrepressor.
lacI supperrepressor can not bind inducer.
lacIs mutant encodes a diffusible element that binds to operator regardless of
chromosomal location (trans acting element).
Copyright © 2010 Pearson Education, Inc.
Question
• For the lac genotypes shown in the accompanying table, predict
whether the structural gene (Z) is constitutive, permanently
repressed, or inducible in the presence of lactose.
Genotype
I+O+Z+
I-O+Z+
I+OCZ+
I-O+Z+/F’I+
I+OCZ+/F’O+
IsO+Z+
IsO+Z+/F’I+
Constitutive
Repressed
Inducible
Question
•
For the genotypes and conditions (lactose present or absent) shown in the
accompanying table, predict whether functional enzymes (F), nonfunctional
enzymes (NF), or no enzymes (NO) are made.
Genotype
Conditions
I+O+Z+
No Lactose
I+OCZ+
Lactose
I-O+Z-
No Lactose
I-O+Z-
Lactose
I-O+Z+/F’I+
No Lactose
I+OCZ+/F’O+
Lactose
I+O+Z-/F’I+O+Z+
Lactose
I-O+Z-/F’I+O+Z+
No Lactose
ISO+Z+/F’O+
No Lactose
I+OCZ+/F’O+Z+
Lactose
F
NF
NO
Positive Control Increases Transcription
of lacZ, lacY, and lacA
• cAMP binds to CRP
(cAMP receptor protein)
when glucose is low.
• CRP binds to regulatory
region.
• Enhances activity of RNA
polymerase at the lac
promoter.
Positive Regulation
The formation of cAMP from ATP, catalyzed by adenyl cyclase.
In the absence of glucose, cAMP levels increase, resulting in the
formation of a CAP-cAMP complex, which binds to the CAP site of the
promoter, stimulating transcription.
In the presence of glucose, cAMP levels decrease, CAP-cAMP complexes
are not formed, and transcription is not stimulated.
How Regulatory Proteins Interact with
RNA Polymerase
• Negative regulators (lac repressor)
– Physically block DNA-binding sites of RNA
polymerase.
• Positive regulators
• Establish physical contact with RNA polymerase
enhancing enzyme’s ability to initiate transcription.
Summary of Gene Regulation in lac Operon
• The trp operon is an example of a repressible operon,
one that is inhibited when a specific small molecule
binds allosterically to a regulatory protein.
• In contrast, an inducible operon is stimulated when
a specific small molecule interacts with a regulatory
protein.
– In inducible operons, the regulatory protein is active
(inhibitory) as synthesized, and the operon is off.
– Allosteric binding by an inducer molecule makes the
regulatory protein inactive, and the operon is on.
Biosynthesis of Trp
trp Operon
The components involved in the regulation of the tryptophan operon.
Trp Operator Leader RNA
(a) The trp operon, showing the leader region, followed by the five trp
genes.
(b) The leader region in the mRNA is enlarged to show the translation
start codon (AUG), the two tryptophan codons (UGGUGG), and the
string of U residues at the end of the leader. The four regions
marked 1, 2, 3, and 4 indicate the four sequence regions that have
potential to form stems by base pairing.
In the absence of tryptophan, an inactive repressor is made that cannot
bind to the operator (O), thus allowing transcription to proceed.
In the presence of tryptophan, it binds to the repressor, causing an
allosteric transition to occur. This complex binds to the operator region,
leading to repression of the operon.
The Attenuation of Gene Expression: Fine
Tuning of the trp Operon through
Termination of Transcription
• The presence of tryptophan activates a repressor of
the trp operon
– trpR gene produces repressor.
– Corepressor : tryptophan binds to trp repressor
allowing it to bind to operator DNA and inhibit
transcription.
Attenuation in the trp Operon
Attenuator
λ Phage Life Cycle
▶ Lytic Cycle: Cro
▶ Lysogenic Cycle: CI
λ Phage Map
Control Region of phage λ
□ PR and PL are strong, constitutive promoter.
□ PRM is a weak promoter and only directs efficient
transcription when an activator is bound.
Transcriptional Control in Lytic and
Lysogenic Growth
Relative Positions of
Promoters and Operators
► Upon infection, transcription is immediately initiated
from the two constitutive promoters PR and PL.
► PR directs synthesis of both Cro and CII.
► Cro expression favors lytic cycle.
► When Cro reaches a certain level, it will bind OR3 and
block PRM.
► CII expression favors lysogenic growth by directing
transcription of the repressor gene.
► For successful lysogeny, repressor must then bind to OR1
and OR2 and activate PRM before Cro can inhibit that
promoter.
Cooperative Binding of λ Repressor
► CI: OR1>OR2, OR3
► Cro: OR3>OR2, OR1
Action of λ Repressor and Cro
Antiterminator
Genes and Promoters Involved in the
Lytic/Lysogenic Choice
Establishment of Lysogeny
• If growth is good, FtsH (protease) is very active, CII is
destroyed efficiently, repressor is not made, and phage
tend to grow lytically.
• In poor growth conditions, the opposite happens: low
FtsH activity, slow degradation of CII, repressor
accumulation, and a tendency toward lysogenic
development.
• Levels of CII are also modulated by the phage protein
CIII.
• CIII stabilizes CII, probably because it acts as an
alternative substrate for FtsH.
Gene Regulation
in Eukaryotes
In Eukaryotes Three RNA Polymerases
Transcribe Different Sets of Genes
• RNA polymerase I
transcribes rRNA.
– rRNAs are made of
tandem repeats on one
or more chromosomes.
– RNA polymerase I
transcribes one primary
transcript which is
broken down into 28S
and 5.8S by processing.
• RNA polymerase III transcribes tRNAs and other small
RNAs (5s rRNA, snRNAs).
• RNA polymerase II recognizes cis-acting regulatory regions
composed of one promoter and one or more enhancers.
– Promoter contains initiation site and TATA box.
– Enhancers are distant from target gene.
• Sometimes called upstream activation sites (UAS).
• RNA polymerase II transcribes all protein coding genes.
– Primary transcripts are processed by splicing, a poly A tail is
added to the 3’ end, and a 5’ GTP cap is added.
trans-acting Proteins Control
Transcription from Class II Promoters
• Basal factors bind to the
promoter.
– TBP : TATA box binding
protein
– TAF : TBP associated
factors
• RNA polymerase II binds to
basal factors.
Assembly of
General
Transcription
Factors
Copyright © 2010 Pearson Education, Inc.
Activator Proteins
• Also called transcription factors.
• Bind to enhancer DNA in specific ways.
• Interact with other proteins to activate and increase
transcription as much as 100-fold above basal levels.
• Two structural domains mediate these functions.
– DNA-binding domain
– Transcription-activator domain
• Transcriptional
activators bind to
specific enhancers at
specific times to
increase transcriptional
levels.
Formation of DNA Loop
Copyright © 2010 Pearson Education, Inc.
Some Proteins Affect Transcription
without Binding to DNA
• Coactivator : binds to and affects activator protein
which binds to DNA.
• Enhancerosome : multimeric complex of proteins
– Activators
– Coactivators
– Repressors
– Corepressors
Repressors
• Reduction of transcriptional activation but do not
affect basal level of transcription.
– Activator-repressor competition
– Quenching (corepressors)
• Some repressors stop basal level of transcription.
– Binding directly to promoter
– Bind to DNA sequences farther from promoter,
contact basal factor complex at promoter by
bending DNA causing a loop where RNA
polymerase can not access the promoter.
Transcription Factors may Act as Activators
or Repressors or Have No Affect
• Action of transcription factor depends on
– Cell type
– Gene it is regulating
Other Mechanisms of Gene Regulation
• Chromatin structure
– Slows transcription
– Hypercondensation stops transcription
• Genomic imprinting
– Silences transcription selectively if inherited from one parent
• Some genes are regulated after transcription
– RNA splicing can regulate expression
– RNA stability controls amount of gene product
– mRNA editing can affect biological properties of protein
– Noncoding sequences in mRNA can modulate translation
– Protein modification after translation can control gene
function
Normal Chromatin Structure Slows
Transcription
Remodeling of Chromatin Mediates
the Activation of Transcription
Copyright © 2010 Pearson Education, Inc.
Hypercondensation over Chromatin
Domains Causes Transcriptional Silencing
Noncoding RNAs Play Multiple Roles
in Controlling Gene Expression
• Only a small fraction of DNA codes for proteins, rRNA,
and tRNA.
• A significant amount of the genome may be transcribed
into noncoding RNAs.
• Noncoding RNAs regulate gene expression at two
points: mRNA translation and chromatin configuration.
MicroRNAs (miRNAs) and Small
Interfering RNAs (siRNAs)
• miRNAs
– 21-25 nt, hairpin (stem-loop) structure.
• siRNAs
– 20-25 nt, dsRNA.
Copyright © 2010 Pearson Education, Inc.
Effects on mRNAs by MicroRNAs
(miRNAs) and Small Interfering RNAs
(siRNAs)
• MicroRNAs (miRNAs) are small single-stranded RNA
molecules that can bind to mRNA.
• These can degrade mRNA or block its translation.
Hairpin
miRNA
Hydrogen
bond
Dicer
miRNA
5′ 3′
(a) Primary miRNA transcript
mRNA degraded
miRNAprotein
complex
Translation blocked
(b) Generation and function of miRNAs
• The phenomenon of inhibition of gene expression by
RNA molecules is called RNA interference (RNAi).
• RNAi is caused by small interfering RNAs (siRNAs).
• siRNAs and miRNAs are similar but form from different
RNA precursors.
The Nobel Prize in Physiology or
Medicine 2006
"for their discovery of RNA interference –
gene silencing by double-stranded RNA"
Mechanism of Action of RNAi