why genes are regulated? minimize energy consumption--why express a gene you do not need? (economy)...

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 transcriptionA 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

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

The outcome of regulation may be positive or negative.

tran

s-ac

ting

ele

men

t

(cis-acting element: usually upstream of target genes including promoter and terminator.)

structural gene

regulatorgene

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

Components in regulatory circuits

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

Regulators and mechanisms:

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 anotherAutogenous: a protein regulates expression of the gene that codes for itself

Negative control Positive controlHalf of prokaryotic genes Half of prokaryotic genes

Most of eukaryotic genes

A diffusible trans-acting factor bound to cis-acting targeting site(s) is:

default state of genes is inactivedefault state of genes is active

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

• Three genes are coordinately regulated.

• Controlled by the metabolic state of the cell -- carbon source.– Negative control

• lac I gene

– Positive control• CAP-cAMP

By Monod,1940

• 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.

Lac z Lac y Lac aP/OLac I

The Lac operon

Element purpose

Operator (LacO)

binding site for repressor

Promoter (LacP)

binding site for RNA polymerase

Repressor (LacI)

gene encoding lac repressor protein

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.

Operator Structural gene(s) (lacZYA)

trans-acting factor

+

Cluster is transcribed into a single polycistronic mRNA from a promoter where initiation of transcription is regulated.

tetramer of ~500kD

Cleave the β-galactoside into component sugars.Lactose → glucose + galactose

30kD membrane bound protein

Transports β-galactoside into the cell

Transfer an acetyl group form acetyl-CoA to β-galactosides

tetramer (38kD/each)

10 tetramers in a wild type cell

T T

(independent transcription unit:monocistron)

Operon =

The Lac operon

Element purpose

Operator (LacO)

binding site for repressor

Promoter (LacP)

binding site for RNA polymerase

Repressor (LacI)

gene encoding lac repressor protein

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.

(~26bp)

(~60bp)

Hence, the transcription of genes are turned off by the Lac repressor binding to Olac.

A mutation that inactivates the regulator causes the structural genes to remain in the expressed condition.

(Plac)

(Olac)RNA polymerase Repressor

Repressor and inducer Action

• In absence of lactose, repressor binds DNA and blocks expression of Lac ZYA genes

Lac z Lac y Lac a

LacI gene product bound

+ lactose

Lac z Lac y Lac a

Repressor no longer binds - genes can be active.

LacI released

• In presence of lactose, repressor releases from DNA and lac zya genes express

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)

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

unstable mRNA with ~3min half-life

Protein is more stable

Rapid

indu

ctio

nRapid reverse

(to ensure a minimal amount to start the induction)

IPTG: a gratuitous inducer

• Artificial inducer of the beta galactosidase gene.

• Not metabolized.

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.

The induction does not depend on the activity of inducer.

vs. Lactose

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

1

2

DNA binding preventing Tx

Inducer binding changing shape

1. Sequential expression2. Relative same amount

coordinate regulation

polycistron

Mutagenesis is an approach to analyze the operatorcis-acting mutations: map on promoter and operatortrans-acting product mutations: lacI locus

Un-inducible mutantsconstitutive mutants vs.

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.

Oc type: operator loses binding with lacI lacI- type: loss of function of lacI

Recessive mutation: complementary by wild type

Plac mutant---

lacIs mutant (locked in to the active form that recognizes the operator and prvents transcription.)

Absence the inducer binding site

Promoter loses binding with RNA pol

Absence the DNA binding activity

Allosteric

lacI-d mutant provides the multimeric property of the LacI protein

lacI-d mutant: damages in DNA binding site

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.

negative complementation

(as a dominant negative mutant to LacI its own function)

WT

lacI-

WT

Oc

complementary

Non-complementary

WT

lacIs

WT

lacI-d

Molecular Mechanism of repress working on its operator

: palindorme

Cover by repressor (DNase fingerprint)

Contact (modification)

Functional important bases (essential specific contacts) point mutation

Symmetry in the protein. The operator makesthe same pattern of contacts with a repressor monomer.

Structure of LacI repressor Several domains: N-terminal DNA-binding domain (a.a. 1-59)

a hinge 2X core domains

Headpiece (aa 1-59)independent from core

Fit into the major groove of DNA, make special contacts

Conformation change (from core domains) leads to signal the DNA binding capacity.

6X

: Cleft between core domains

: contains 2X leucine heptad repeats

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.

inducer boundHeadpiece changes its orientationLoses contact with DNA

lacIs

lacI-

Hydrophobic core

Inducer-binding cleft

C-terminal helices

: form dimer

: form tetramer

dimer form

tetramer form

dimer form

inducer binding

form dimer

Higher order of LacI repressor

lacI-d :DNA binding

Why tetramer?Tetramer can bind two operators simultaneously.

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).

O1: in the initial region of the lac operon, strongest affinity for repressorO2: 410bp downstream of startpoint, weaker affinity for repressorO3: 83bp upstream of startpoint, weaker affinity for repressor

O1 O2 X O3 X

2-4X2-4X 100X

CAP

Rep

ress

or t

etra

mer

enable transcription to begin immediately upon induction, instead of waiting for an RNA polymerase to be captured.

CAP (catabolite associated protein)

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 107× 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 104× 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.

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%

Repressor binds to the operator by moving from a low-affinity site rather than by equilibrating from soluation.

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

All of repressors are bound to DNA

Inducer binding leads to lose specificity of bound to operator comparing to other DNA sequences

Re-distribution randomly on the genome

Specificity to the high affinity site

How inducer binding to free repressor?

1. Upset equilibrium 2. Directly displacement (affinity change/flow)

Free repressor binding to DNA results from the reduction of its affinity.

(involves conformational change, but not bond breaking)

How the repressor tetramer set off from the DNA?

(unbalance)

>15 mins

Hence, prefer

Not reversible

Dynamic balance

fast

See next slide

Repression can occur at multiple loci A repressor will act on all loci that have a copy of its target operator sequence

Negative control Positive controlHalf of prokaryotic genes Half of prokaryotic genes

Most of eukaryotic genes

A diffusible trans-acting factor bound to cis-acting targeting site(s) is:

default state of genes is inactivedefault state of genes is active

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 Repression- positive 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.

Glucose repression controls use of carbon sources:

1. E. coli uses glucose in preference to other carbon sources2. Glucose prevents uptake of alternative carbon sources 3. Exclude expression of the operons coding for the enzymes that metabolize other carbon sources (such as lac, gal, ara)

How do bacteria control the carbon sources?

Phenomenon:

Mechanism:

1. Inducer exclusion2. Inhibition of positive regulator, CRP activity (see next)

IIAglcIIAglc-P IIAglc

Phoenolpyruvate:glycose phosphotransferase = PTS

(crr gene)

Regulation of CRP activityCRP 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

IIAglc

Adenylate cyclase

cAMP

-P

Glucose in reducing cyclic AMP levels is to deprive the relevant operons of a control factor necessary for their expression

a positive regulator which may overcome a deficiency in the promoter, e.g. a poor consensus sequence at -35 or -10

IIA-P

Catabolite activator protein (CAP; also known as cAMP receptor protein, CRP)

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.

Catabolite activator protein (CAP) = cAMP receptor protein, CRP)

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.

22.5KDa protein to form a homodimer.

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

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 thestartpoint in the various operons that it regulates

Strong binding Weak binding

How?

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

Class I CAP-dependent promoter activation: CAP dimer interacting with the aCTD of RNAP, which is also comprised of b and s subunits

Class II CAP-dependent promoter activation: CAP dimer interacting with the aCTD and aNTD of RNAP

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),

How CAP work? 3 classes of CAP-dependent promoters

e.g. lac promoter

promoter closed complex formation

promoter closed complex formation

promoter complex to an open complex

e.g. galP1 promoter

1. involve class I and class II mechanisms of action in an additive manner

2. two CAP dimers could function differently

e.g. malK promoter

CRP bends DNA~90°

Diverse control circuits by regulatorsdefault state → expressed default state X expressed

Lac operon

(need a repressor to switch off)interfering

(need an activator to switch on)essential

Trp operon

CRP, σ

1. Allosteric changes2. Activation of proteins (e.g. by Oxidation)3. (de)phosphorylation

via

via

A fail-safe, selective advantage due to increased efficiency (basal level expression)

Outcome is expressed

Outcome isnot expressed

presence of inducer

co-repressor

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.

10~20X tRNA+rRNA ↓3X mRNA ↓~5~10% total RNAs ↓Protein degradation ↑

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.

(Idling reaction)

AP

Stringent response

(Relaxed)

~20secReversed reapidly

e.g. EF-TuEF-G

L11/S50via conformation change

Uncharged tRNA/Ribosome

(p)ppGpp inhibits transcription of rRNA1. Initiation of Tx is specifically inhibited at the promoters of operons coding for rRNA2. 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 indicator2. drives the initiation by stabilizing the

open complex

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

Components in regulatory circuits

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

Regulators and mechanisms:

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 anotherCircuits:

Antagonize: a series of regulators each of which antagonizes anotherAutogenous: a protein regulates expression of the gene that codes for itself

Attenuation: a negative controlalternative secondary structures control attenuation

(analogous to allosteric changes of conformation)

= attenuator

Nucleic acid shifts to different conformation

AttenuationRNAi

External event (signal) controls the formationof the hairpin needed for intrinsic termination.

An intrinsic protein or it may respond to a small molecule (~ repressor or co-repressor) to stabilize or destabilize the hairpin

trp operon in Bacillus subtilisA 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

Uncharged tRNA

TRAP: 11 subunits, bound with tRNAtrp and then wounded by RNA. Response to trp level

anti-TRAP

The trp operon in E. coli

attenuator

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

conformation change of attenuator

→disrupt the loop1 and 2 base pairing

No translation

No termination, transcription continues

Attenuator stop transcription

signal→ ribosome movement

Translated into protein

Mutations increaseexpression 10X in presence of Trp.

Promoter/Operator region

Architecture of trp operon in E. coli: Two folds regulation

intrinsic terminator

Tryptophan dependent translation (movement)

chorismic acid → tryptophan

TrpR: repressor (encoded by another gene)

1

2Regulation of trp genes expression by 2 folds

attenuation

Discover:

independent

140bp

Both respond to the Trp level inside of a cell

X10

X70

The position of the ribosome on mRNA controls attenuation (I): Tryptophan dependent translation (movement)What signal ?

Loop 1 and 2 base pairing impaired Loop2 and 3 forms pairing

Loop 1 and 2 base pairing impairedLoop 2 and 3 base pairing impaired (ribosome continues move)Loop 3 and 4 forms base pairing

ribosome stalling

Leader peptide translation

The position of the ribosome on mRNA controls attenuation (II)

The position of the ribosome on mRNA controls attenuation (III)

Base pairing between loops

1234

loop

cannot translate, ribosome stalled, then disrupts terminator

Translation a leader peptide, termination happens at hairpin

tRNAtrp directly controls the attenuationSignal molecule

Translation can be regulated

1. A repressor protein can regulate translation by preventing a ribosome from binding to an initiation codon.

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. 1.

2.

Even though transcription is coordinated (i.e. operons), the differences can be created in translation

2 cistron

1 cistron

1 and 2 are not mutually exclusive

A feature of protein synthetic apparatus.

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.

Features:Equimolar amountIntermingling Ribosomal proteins, synthesis factors, RNA pol (represent by single gene)Small number of operonsAutonomously regulated: Autogenous regulation

Clusters/operons of gene expression apparatus:Ribosomal proteins, protein-synthesis factors, RNA Pol and factors…

str

spc

S10

α

L11

rif

R-proteins binding affinity: rRNA >mRNA

negative

Phage T4 p32 is controlled by an autogenous circuit p32 binds to its own mRNA to prevent initiation of translation

Each regulatory interaction is unique

A quantitative regulation

Contributes to recombination, repair, replication

When the function of the proteins prevented, more of it is made. 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.

Tubulin mRNA on polysomes is degraded while free tubulin bound

Free tubulin binds on mRNA or nacent peptide

Pool of free tubulins is sensed

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.

In an artificial way to inhibit a mRNA activity by a synthetic antisense RNA, the antisense RNA is needed to a considerable excessive amount.

Formation is happened in either nucleus or cytoplasm

Provide an importance of the time of gene expression

RNA interfering happens in dsRNAs

Complementary base pairing to control RNA activity by another RNA

Base pairing

Usually, regulator RNAs are small (short) RNA molecules (single-stranded)

(usually, targeted RNAs are mRNAs)

Trans-acting factor

complementary

A double stranded region

In vivo

General mechanisms: changes in 2° structure of the target

1. 2.

Forming a duplex region → block initiation of translation (1)cause termination of transcription (3)create a target for an endonuclease (2)

3.

(No allosteric affecting by small mols)

Bacterial oxyS RNA is a regulator RNA (sRNA)shortOne of 17 different sRNAs, which affects many targets by repression or activation.

Respond to oxidation by expressing antioxidant defense genes.

Not a protein but a short RNA

A trans-acting regulator at post-transcription levels; >10 target genes

initiation site

Repress ribosome binding Prevent translation

The oxyR mutant is resulted from the overxpression of the oxyS sRNA regulator

encodes a protein that is believed to be part of the export apparatus for flagellum assembly

OxyS rpoS

RprA DsrA

FlhA

Global sRNA regulator in bacteria

Hfq protein

RNA binding protein; needed for Qβphage replicationSimilar to Sm protein in eukaryotes (binds to snRNA)

Change

2 str

ucture

of b

ound R

NAs

Sigma factor for general stress response

encodes a protein that is believed to be part of the export apparatus for flagellum assembly

Improve the ss exposure

Discovery of MicroRNAsRegulate 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

In eukaryotes

Post-transcriptional

transcriptional

Imprecise base pairing by miRNA (21 bases)

Lin4 miRNA regulates the expression of stage-specific developmental eventsInduces late event of larva development

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

Recovery in tobacco plants infected with tobacco ringspot virus.The original legend1 to the figure reads ‘Turkish tobacco plant 23 daysafter 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

RNA interference (RNAi) and gene silencingtriggers 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)

dicer

RISC (RNA-induced silencing complex)

Mechanism of RNAi

The general pathway of RNAi in vitro

Small RNA biogenesis in animals

~70nt

~21-25nt

Pasha (DGCR8)

A mRNA transcript by RNA Pol II

histone methyltransferases

Small RNAs act upstream of several effectors

The potential of RNAi in studies of gene function and genomics

Post-transcription gene silencing (PTGS): dsRNA inhibits expression of a gene existsin plants and fungi.

virus-induced gene silence (VIGS) is a natural defense mechanism in plants. (during virus replicating which forms dsRNA intermediates; required RNA-dependent RNA polymerase)

Viral infection:

siRNA or signal can be systemically transported to other non-infected cells

amplification

Virus as a vector to harbor exogenous gene fragments.Inoculation of the recombinant virus into cells, the corresponding endogenousgenes are silenced

Virus as a vector for gene silencing

RNAi has been observed in plants, fungi, mammals, worms, and flies and offers significant therapeutic potential.(a) RNAi as a natural process. (b) RNAi using synthetic siRNAs.

The mechanism of RNAi