Download Why genes are regulated?

Why genes are regulated?
• Minimize energy consumption--why express a
gene you do not need? (economy)
• Control growth--many cells in a mature
organism do not grow, and expression of
genes involved in promoting cell division is
tightly regulated. (physiological balance)
• Development--inappropriate expression of
genes that regulate differentiation may
adversely affect development (pathology)
• Response to environment (dynamic)
How is gene regulation controlled?
Transcription- at initiation and at termination.
Less happened at elongation.
RNA processing- only happened in Eukaryotes
via modification, splicing, transport, or stability.
not available in Prokaryotes (overcome by
transcription is intimately tied up with translation)
Translation- its regulation is analogous to those of transcription,
happened at initiation and at termination
Gene regulation in transcription
A principle example by Jacob and Monod (1961)
cis-acting element: not convertible, function as a DNA sequence in situ,
phyiscal linked
trans-acting element: diffusible
trans-acting element
Gene activity is regulated by the specific interactions of the trans-acting products
(usually proteins/RNAs) with the cis-acting sequences (usually sites in DNA).
diffuse
structural gene
regulator
gene
(cis-acting element: usually upstream of target genes
including promoter and terminator.)
The outcome of regulation may be positive or negative.
Components in regulatory circuits
Regulators and mechanisms:
Protein regulators:1. allostery
- two different sites,
one for nucleic acid target, the other for a small molecule
2. multimer (usually has a symmetrical organization)
-cooperative binding effect
RNA regulators: usually a small RNA molecule
1. changes in 2° structure
2. complementary base pairing
Consequence of targeting:
a. Formation of the double helical structure may itself be sufficient.
b. Duplex formation may be important because it sequesters a region of
the target RNA
The relationship of regulators in gene regulatory circuits:
Coordinate: an operator controls the expression of many genes
Network: one regulator is required for the production of another (cascade)
Antagonize: a series of regulators each of which antagonizes another
Autogenous: a protein regulates expression of the gene that codes for itself
A diffusible trans-acting factor bound to cis-acting targeting site(s) is:
Negative control
Half of prokaryotic genes
Positive control
default state of genes is active
Half of prokaryotic genes default state of genes is inactive
Most of eukaryotic genes
activators
control level
Repressors and activators are required very short cis-acting sequences (<10bp) to function.
Such as the hexamers of -35 and -10 for the RNA polymerase.
Bacterial Lactose Operon
Lac I P/O
Lac z
Lac y
Lac a
By Monod,1940
• Three genes are
coordinately regulated.
• Controlled by the
metabolic state of the
cell -- carbon source.
– Negative control
• lac I gene
– Positive control
• CAP-cAMP
•
•
•
•
•
Jacob & Monod
Induction of the β- galactosidase gene in
response to lactose.
Lactose - Inducer of activity
Synthesis of new protein.
In the absence of lactose, the gene is not
expressed.
The Lac operon
Element
purpose
Operator
(LacO)
binding site for repressor
Promote
r (LacP)
binding site for RNA polymerase
gene encoding lac repressor protein
Repress
or (LacI)
Binds to DNA at operator and blocks binding of
RNA polymerase at promoter
Pi
promoter for LacI
CAP
binding site for cAMP/CAP complex
The lac operon- A negative control
(Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor )
Polycistron– bacterial structural genes are often organized into clusters, coordinately controlled by
means of interactions at a single promoter.
Monocistron– only one gene is controlled by an individual promoter.
Operon = Operator +
Structural gene(s) (lacZYA)
T
trans-acting factor
tetramer (38kD/each)
10 tetramers in a wild type cell
(independent transcription unit:
monocistron)
T
tetramer of ~500kD
Cleave the β-galactoside into
component sugars.
Lactose → glucose + galactose
30kD membrane
bound protein
Transfer an acetyl group form
acetyl-CoA to β-galactosides
Transports β-galactoside
into the cell
Cluster is transcribed into a single polycistronic mRNA from a promoter where
initiation of transcription is regulated.
The Lac operon
Element
purpose
Operator
(LacO)
binding site for repressor
Promote
r (LacP)
binding site for RNA polymerase
gene encoding lac repressor protein
Repress
or (LacI)
Binds to DNA at operator and blocks binding of
RNA polymerase at promoter
Pi
promoter for LacI
CAP
binding site for cAMP/CAP complex
Metabolic action of LacZ gene product, β-galactosidase
LacI repressor form a tetramer bound onto the operator
How the lac genes are controlled?
Repressor and RNA polymerase bind at sites that overlap around
the transcription startpoint of the lac operon.
Hence, the transcription of genes are turned off by the Lac repressor binding to Olac.
RNA polymerase
(Plac)
Repressor
(~60bp)
(Olac)
(~26bp)
A mutation that inactivates the regulator causes the structural genes to remain in the expressed condition.
Repressor and inducer Action
• In absence of lactose, repressor binds DNA and blocks
expression of Lac ZYA genes
LacI gene product bound
Lac z
Lac y
• In presence of lactose, repressor releases
from DNA and lac zya genes express
LacI released
Lac z
Lac y
Lac a
+ lactose
Lac a
Repressor no longer binds - genes can be active.
Induction of lac operon
Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor
The expression of lac operon: an induction
Induction of mRNA and protein
(also happened in yeast)
unstable mRNA with ~3min half-life
Protein is more stable
Within 2-3 mins, ~5000 enzyme molecules
are present and can reach up to 5%-10% of
the total soluble protein of the bacterium.
~5 molecules (to
ensure a minimal amount to start the induction)
IPTG: a gratuitous inducer
• Artificial inducer of the
beta galactosidase
gene.
• Not metabolized.
vs.
Lactose
The induction does not depend on the activity of inducer.
The system must possess some component, distinct from the target enzyme, that recognizes the appropriate substrate;
and its ability to recognize related potential substrates is different from that of the enzyme.
Conversion of repressors into an inactive
leads to gene expression: an allosteric control
LacI repressor possesses dual properties: 1. binds to DNA preventing transcription
(Allosteric effect) 2. interacts with small-molecule inducer changing its own conformation
2
Inducer binding
changing shape
1
DNA binding preventing Tx
polycistron
coordinate
regulation
1. Sequential expression
2. Relative same amount
Mutagenesis is an approach to analyze the operator
Un-inducible mutants
constitutive mutants
vs.
cis-acting mutations: map on promoter and operator
trans-acting product mutations: lacI locus
Oc type: operator loses binding with lacI
lacI- type: loss of function of lacI
Absence the DNA
binding activity
Allosteric
Recessive mutation: complementary by wild type
cis-dominance: 1.mutation(s) at any site that is physically
contiguous with the sequences it controls
2. cannot be assigned to a complementation group.
Consistent with the operator as a typical cis-acting site, whose
function depends upon recognition of its DNA sequence by
some trans-acting factor.
Plac mutant--Promoter loses binding
with RNA pol
Absence the inducer
binding site
lacIs mutant
(locked in to the active form that recognizes
the operator and prvents transcription.)
lacI-d mutant provides the multimeric property of the LacI protein
lacI-d mutant: damages in DNA binding site
(as a dominant negative mutant to LacI its own function)
negative complementation
Tetramer is formed in LacI repressor. Heterotetramer may be formed as interallelic complemenatation.
Combination between lacI-d and lacI+ leads to occur negative complementation,
suggesting lacI-d is called dominant negative.
WT
lacI-
complementary
WT
Oc
Noncomplementary
WT
lacIs
WT
lacI-d
Molecular Mechanism of repress working on its operator
: palindorme
Symmetry in
the protein.
The operator makes
the same pattern of
contacts with a repressor
monomer.
Functional important bases
(essential specific contacts) point mutation
Contact (modification)
Cover by repressor
(DNase fingerprint)
Structure of LacI repressor
Several domains: N-terminal DNA-binding domain (a.a. 1-59)
a hinge
2X core domains
Fit into the major groove of DNA, make special contacts
Conformation change (from core domains)
leads to signal the DNA binding capacity.
Headpiece (aa 1-59)
independent from core
6X
: Cleft between core domains
Headpiece changes its orientation
Loses contact with DNA
: contains 2X leucine heptad repeats
inducer bound
A half-site of the dyad symmetry sequence can bind an
intact repressor monomer.
The affinity for DNA is many orders of magnitude
higher by intact repressor, that is dimer/tetramer.
Higher order of LacI repressor
form dimer
Inducer-binding cleft
Hydrophobic core
: form dimer
C-terminal helices
: form tetramer
dimer form
lacIs
inducer binding
dimer form
lacItetramer form
lacI-d
:DNA binding
Why tetramer?
Tetramer can bind two operators simultaneously.
O1: in the initial region of the lac operon, strongest affinity for repressor
O2: 410bp downstream of startpoint, weaker affinity for repressor
O3: 83bp upstream of startpoint, weaker affinity for repressor
O2 X
O3 X
CAP (catabolite associated protein)
2-4X
2-4X
100X
Repressor tetramer
O1
CAP
In fact, the repressor binding onto operator(s) enhances RNA polymerase binding at the promoter. However the bound RNA
polymerase is prevented from initiating transcription (stored at closed complex).
enable transcription to begin immediately upon induction, instead of waiting for an RNA polymerase to be captured.
Repressor is always bound to DNA
Proteins that have a high affinity for a specific DNA sequence also have a low affinity for other DNA sequences.
Every base pair in the bacterial genome is the start of a low-affinity binding-site for repressor.
The large number of low-affinity sites ensures that all repressor protein is bound to DNA.
Repressor binds to the operator by moving from a low-affinity site rather than by equilibrating from solution.
The operator competes with low-affinity sites to bind repressor
In the absence of inducer, the operator has an affinity for repressor that is 10 7× that of a low affinity site.
The level of 10 repressor tetramers per cell ensures that the operator is bound by repressor 96% of the time.
Induction reduces the affinity for the operator to 10 4× that of low-affinity sites, so that only 3% of operators are bound.
Induction causes repressor to move from the operator to a low-affinity site by direct displacement.
These parameters could be changed by a reduction in the effective concentration of DNA in vivo.
A kinetic view of repressors on an operator
What affects the repressor binding to the operator:
genome size, specificity of the repressor, the amount of the repressor existed/required
1. Repressors have a high affinity for a specific DNA sequence
and also have a low affinity for other DNA sequences.
2. Hence, the large number of low-affinity sites ensures
that all repressors are bound to DNA.
3. Excessive repressor proteins ensure that the operator is occupied
by a repressor at ~96%
All of repressors are bound to DNA
Re-distribution randomly on the genome
Inducer binding leads to lose specificity of bound to
operator comparing to other DNA sequences
Specificity to the high affinity site
Repressor binds to the operator by moving from a
low-affinity site rather than by equilibrating from soluation.
How inducer binding to free repressor?
Free repressor binding to DNA results from the reduction of its affinity.
How the repressor tetramer set off from the DNA?
1. Upset equilibrium 2. Directly displacement (affinity change/flow)
(involves conformational change,
(unbalance)
but not bond breaking)
Dynamic balance
>15 mins
Hence, prefer
fast
See next slide
Not reversible
Repression can occur at multiple loci
A repressor will act on all loci that have a copy of its target operator sequence
A diffusible trans-acting factor bound to cis-acting targeting site(s) is:
Negative control
Half of prokaryotic genes
Positive control
default state of genes is active
Half of prokaryotic genes default state of genes is inactive
Most of eukaryotic genes
activators
control level
Repressors and activators are required very short cis-acting sequences (<10bp) to function.
Such as the hexamers of -35 and -10 for the RNA polymerase.
Catabolite Repressionpositive control
• Additional control mechanism prevents
Lac operon expression when Glucose is
present.
• Lactose + Glucose to E. coli-- Lac operon
will remain off.
• Cells have a glucose sensor.
How do bacteria control the carbon sources?
Phoenolpyruvate:glycose phosphotransferase = PTS
Glucose repression controls use of carbon sources:
Phenomenon:
1.
2.
3.
E. coli uses glucose in preference to other carbon sources
Glucose prevents uptake of alternative carbon sources
Exclude expression of the operons coding for the enzymes
that metabolize other carbon sources (such as lac, gal, ara)
Mechanism:
1. Inducer exclusion
2. Inhibition of positive regulator, CRP activity
(see next)
IIAglc-P
(crr gene)
IIAglc
IIAglc
Regulation of CRP activity
a positive regulator which may overcome a deficiency in the promoter, e.g. a poor consensus sequence at -35 or -10
CRP activator controls the activity of a large set of operons in E. coli.
A dimer of CRP is activated by a single molecules of cyclin AMP
IIA-P
IIAglc
Adenylate
cyclase
cAMP
Catabolite activator protein (CAP; also
known as cAMP receptor protein, CRP)
Glucose in reducing cyclic AMP levels is to deprive
the relevant operons of a control factor necessary for
their expression
-P
Catabolite activator protein (CAP)
= cAMP receptor protein, CRP)
22.5KDa protein to form a homodimer.
an N-terminal domain required for CAP dimerisation and the binding of cAMP,
a C-terminal domain that contains a helix-turn-helix motif required for the binding of DNA.
Gene activator: AR1 (activating region 1) region within the C-terminal domain, which interacts with the C-terminal
domain of the RNAP alpha subunit (aCTD);
AR2 (activating region 2) region within the N-terminal domain, which interacts with the N-terminal
domain of RNAP alpha subunit (aNTD);
AR3 (activating region 3) region within the N-terminal domain, which interacts with the RNAP
sigma70 (s70) subunit.
CAP is one of over 300 transcription factors used by Escherichia coli alone. Such as metabolism of sugars and amino acids,
transport processes, protein folding, toxin production and pilus synthesis.
TGTGA conserved pentamer is essential and an inverted repeat version given the strongest interaction with
CRP
How CRP activator works to positively control transcription?
1. Increase the rate of initial binding to form a closed comolex
2. CAP + cAMP allow formation of an open promoter comoplex
How?
CRP: form a dimer (22.5kD/each),
each of them has a DNA binding region and a transcription-activating region.
Binding ~22bp in a responsive promoter
TGTGA conserved pentamer is essential and an inverted
repeat version given the strongest interaction with CRP
(increase affinity to DAN a lot)
CRP binding sites lie in different locations relative to the
startpoint in the various operons that it regulates
Strong binding
Weak binding
CRP in regulation of lac operon
Only the activating region of the subunit nearer the startpoint is required,
presumably because it touches RNA polymerase.
>>>> Orientation-independent
Dimer promotes the binding affinity of CAP onto DNA
The CRP protein can bind at different sites relative to RNA poymerase
How CAP work? 3 classes of CAP-dependent promoters
promoter closed complex formation
e.g. lac promoter
Class I CAP-dependent promoter activation: CAP dimer interacting with the aCTD of RNAP, which is also comprised of b and s subunits
promoter complex to an open complex
e.g. galP1 promoter
promoter closed complex formation
Class II CAP-dependent promoter activation: CAP dimer interacting with the aCTD and aNTD of RNAP
1. involve class I and class II mechanisms of action in an additive manner
e.g. malK promoter
2. two CAP dimers could function differently
Class III CAP-dependent promoter activation: two CAP dimers interacting with the aCTD of RNAP
Journal of Molecular Biology 293, S. Busby and R. Ebright, Transcription Activation by Catabolite Activator Protein (CAP), 199-213 (1999),
CRP bends DNA
~90°
Diverse control circuits by regulators
default state → expressed
(need a repressor to switch off)
interfering
Lac operon
default state X expressed
(need an activator to switch on)
essential
CRP, σ
presence of
inducer
via
1. Allosteric changes
2. Activation of proteins
(e.g. by Oxidation)
3. (de)phosphorylation
Outcome is
expressed
Trp operon
via
co-repressor
Outcome is
not expressed
A fail-safe, selective advantage due to
increased efficiency (basal level expression)
The stringent response produces (p)ppGpp (alarmones)
Poor growth conditions cause bacteria to produce the small molecule regulators
ppGpp and pppGpp to shut down a wide range of activities associated with inhibition of Tx.
Stringent response
L11/S50
via conformation
change
Uncharged tRNA
/Ribosome
10~20X tRNA+rRNA ↓
3X mRNA ↓
~5~10% total RNAs ↓
Protein degradation ↑
(Relaxed)
e.g. EF-Tu
EF-G
~20sec
Reversed reapidly
(Idling reaction)
The stringent factor RelA is a (p)ppGpp
synthetase that is associated with ~5% of
ribosomes.
RelA is activated when the A site is occupied by
an uncharged tRNA.
One (p)ppGpp is produced every time an
uncharged tRNA enters the A site.
P
A
(p)ppGpp inhibits transcription of rRNA
1.
2.
Initiation of Tx is specifically inhibited at the promoters of operons coding for rRNA
The elongation phase of Tx of many or most templates is reduced by ppGpp
The level of protein synthesis increases in proportion with
the growth rate.
Ribosome ≡ protein synthesis ≡ cell growth
NTP level as 1. an indicator
2. drives the initiation by stabilizing the
open complex
Components in regulatory circuits
Regulators and mechanisms:
Protein regulators:1. allostery
- two different sites,
one for nucleic acid target, the other for a small molecule
2. multimer (usually has a symmetrical organization)
-cooperative binding effect
RNA regulators: usually a small RNA molecule
1. changes in 2° structure
2. complementary base pairing
Consequence of targeting:
a. Formation of the double helical structure may itself be sufficient.
b. Duplex formation may be important because it sequesters a region of
the target RNA
The relationship of regulators in gene regulated:
Coordinate (operon): an operator controls the expression of many genes
Network (cascade): one regulator is required for the production of another
Circuits:
Antagonize: a series of regulators each of which antagonizes another
Autogenous: a protein regulates expression of the gene that codes for itself
Attenuation: a negative control
alternative secondary structures control attenuation
(analogous to allosteric changes of conformation)
External event (signal) controls the formation
of the hairpin needed for intrinsic termination.
= attenuator
An intrinsic protein or it may respond to a
small molecule (~ repressor or co-repressor)
to stabilize or destabilize the hairpin
Nucleic acid shifts to
different conformation
Attenuation
RNAi
trp operon in Bacillus subtilis
A terminator protein , TRAP, is activated by tryptophan to prevent transcription of trp genes.
In the absence of tryptophan, the activity of TRAP is inhibited by uncharged tRNAtrp anti-TRAP.
TRAP: 11 subunits, bound with tRNAtrp
and then wounded by RNA. Response to trp level
TRAP
Uncharged tRNA
anti-TRAP
The trp operon in E. coli
Ribosome (translation) is the intrinsic protein (stimulated by trp) to stabilize/destabilize the
termination hairpin (attenuator) on mRNA.
Anti-termination: (signal dependent)
A leader sequence preceding the trp genes contains an
attenuator (terminator hairpin) whose 2° structure can be
changed dependent on the translation of the leader peptide.
Resided wthin
attenuator
Attenuator stop transcription
No translation
signal→ ribosome
movement
conformation change
of attenuator
→disrupt the loop1 and 2 base pairing
No termination, transcription continues
Architecture of trp operon in E. coli: Two folds regulation
Tryptophan dependent translation (movement)
Promoter/Operator
region
140bp
Translated into protein
Discover:
chorismic acid
→ tryptophan
1 TrpR: repressor X70
Mutations increase
expression 10X in
presence of Trp.
Regulation of trp genes
expression by 2 folds
(encoded by another gene)
independent
2
attenuation
Both respond to the Trp level inside of a cell
X10
intrinsic terminator
The position of the ribosome on mRNA controls attenuation (I)
What signal ? : Tryptophan dependent translation (movement)
ribosome stalling
Loop 1 and 2 base pairing impaired
Loop2 and 3 forms pairing
Leader peptide translation
Loop 1 and 2 base pairing impaired
Loop 2 and 3 base pairing impaired
(ribosome continues move)
Loop 3 and 4 forms base pairing
The position of the ribosome on mRNA controls attenuation (II)
The position of the ribosome on mRNA controls attenuation (III)
Base pairing between loops
loop
1
2
3
4
Signal molecule
tRNAtrp directly controls the attenuation
Translation a leader peptide,
termination happens at hairpin
cannot translate, ribosome stalled,
then disrupts terminator
A feature of protein synthetic apparatus.
Translation can be regulated
Even though transcription is coordinated (i.e. operons), the differences can be created in translation
1. A repressor protein can regulate translation by preventing a ribosome from binding to an initiation codon.
1.
2. Accessibility of initiation codons in a polycistronic mRNA can be controlled by changes in the structure of
the mRNA that occur as the result of translation.
2.
1 cistron
2 cistron
1 and 2 are not mutually exclusive
Proteins that bind to sequences within the initiation regions of mRNAs
may function as translational repressors
r-protein synthesis is controlled by (-) autogenous regulation
Translation of an r-protein operon can be controlled by a product of the operon that binds to a site on the polycistronic mRNA
Clusters/operons of gene expression apparatus:
Ribosomal proteins, protein-synthesis factors, RNA Pol and factors…
R-proteins binding affinity: rRNA >mRNA
str
spc
S10
α
L11
rif
Features:
Equimolar amount
Intermingling Ribosomal proteins, synthesis factors, RNA pol (represent by single gene)
Small number of operons
Autogenous
Autonomously regulated: negative
regulation
Phage T4 p32 is controlled by an autogenous circuit
p32 binds to its own mRNA to prevent initiation of translation
A quantitative regulation
Contributes to recombination,
repair, replication
When the function of the proteins prevented, more of it is made.
Each regulatory interaction is unique
such as RegA proteins
Autogenous regulation is often used to control
synthesis of macromolecular assemblies
The precursor to microtubules, free tubulin protein, inhibits translation of tubulin mRNA.
Pool of free tubulins is sensed
Free tubulin binds on mRNA or nacent peptide
Tubulin mRNA on polysomes is
degraded while free tubulin bound
Extrinsic :repressor regulators
Intrinsic: autogenous controls
RNA regulators
A regulator RNA is a small RNA with a single-stranded region that can
pair with a single-stranded region in a target RNA
Antisense RNA: in both prokaryotes and eukaryotes
A reversing orientation of a gene with regard to its promoter make an antisense RNA.
Provide an importance of the time of gene expression
Formation is happened in either
nucleus or cytoplasm
In an artificial way to inhibit a mRNA activity by a synthetic antisense RNA,
the antisense RNA is needed to a considerable excessive amount.
RNA interfering happens in dsRNAs
In vivo
Complementary base pairing to control RNA activity by another RNA
(usually, targeted RNAs are mRNAs)
Trans-acting factor
A double stranded region
Base pairing
complementary
Usually, regulator RNAs are small (short) RNA molecules (single-stranded)
General mechanisms: changes in 2° structure of the target
(No allosteric affecting by small mols)
Forming a duplex region → block initiation of translation (1)
cause termination of transcription (3)
create a target for an endonuclease (2)
2.
1.
3.
Bacterial oxyS RNA is a regulator RNA (sRNA)
One of 17 different sRNAs, which affects many targets by repression or activation.
Respond to oxidation by expressing antioxidant defense genes.
short
Repress ribosome binding
Prevent translation
encodes a protein that is believed to be part of
the export apparatus for flagellum assembly
initiation site
Not a protein but a short RNA
A trans-acting regulator at post-transcription levels; >10 target genes
The oxyR mutant is resulted from the overxpression of the oxyS sRNA regulator
Global sRNA regulator in bacteria
RNA binding protein;
needed for Qβphage replication
Similar to Sm protein in eukaryotes (binds to snRNA)
Improve the ss exposure
Hfq protein
RprA
OxyS
FlhA
encodes a protein that is believed to be part of
the export apparatus for flagellum assembly
DsrA
rpoS
Sigma factor for general stress response
In eukaryotes
Discovery of MicroRNAs
Regulate gene expression by base pairing with complementary sequences
in target mRNAs
858-62
862-64
Dartmouth Medical School, Department of Genetics,
Hanover, NH
Whitehead Institute for Biomedical Research, and
Department of Biology, Massachusetts Institute of Technology,
9 Cambridge Center, Cambridge, MA
Lin4 miRNA regulates the expression of stage-specific developmental events
Induces late event of larva development
Imprecise base pairing by miRNA (21 bases)
Post-transcriptional
transcriptional
Several target sites are existed close to the 3’ end of target mRNA
(Usually in non-translational region)
Different approaches to study microRNA
MicroRNAs are distributed among eukaryotes
a Nematode pre-miRNA Let 7.
b Arabidopsis pre-miRNA-172.
c Polycistronic pri-miRNA in rice.
d Polycistronic pri-miRNA in nematodes
Structure of pri-miRNAs
alternate poly-A-site
Drosha cleavage sites
Dicer cleavage sites
RNA interference
Recovery in tobacco plants
infected with tobacco ringspot
virus.
The original legend1 to the figure
reads ‘Turkish tobacco plant 23
days
after inoculation with ringspot.
Note the gradual decline in the
development of ringspot
symptoms on the upper leaves
until finally the top leaves appear
perfectly normal’. We now know
that the virus causing the initial
symptoms had activated viral RNA
silencing that inhibited spread of
the infection into the upper leaves,
and caused them to be specifically
immune to tobacco ringspot virus
secondary infection.
RNA interference (RNAi) and gene silencing
triggers degradation of mRNAs complementary to either strand of a short dsRNA,
called siRNA (short interfering RNA), and causes silencing of host genes
: 3’end 2 bases protrusion creates via Dicer enzyme (ATP dependent)
Mechanism of RNAi
dicer
RISC (RNA-induced silencing complex)
The general pathway of RNAi in vitro
Small RNA biogenesis in animals
Pasha (DGCR8)
~21-25nt
~70nt
A mRNA transcript by RNA Pol II
Small RNAs act upstream of several effectors
histone methyltransferases
The potential of RNAi in studies of gene function and genomics
Post-transcription gene silencing (PTGS): dsRNA inhibits expression of a gene exists
in plants and fungi.
Viral infection:
virus-induced gene silence (VIGS) is a natural defense mechanism in plants.
(during virus replicating which forms dsRNA intermediates;
required RNA-dependent RNA polymerase)
amplification
siRNA or signal can be systemically transported to other non-infected cells
Virus as a vector for gene silencing
Virus as a vector to harbor exogenous gene fragments.
Inoculation of the recombinant virus into cells, the corresponding endogenous
genes are silenced
The mechanism of RNAi
RNAi has been observed in plants, fungi, mammals, worms, and flies and offers significant therapeutic potent
(a) RNAi as a natural process. (b) RNAi using synthetic siRNAs.