Prokaryotic Transcription

Transcription

• DNA-dependent RNA synthesis • RNA polymerase doesn’t require a primer • Ribonucleotides not deoxyribonucleotides incorporated

into the polymer • Uracil substituted for thymine • Template for transcription is the antisense strand

Stages

• Initiation: Occurs after promoter recognition and polymerase binding when the first rNTP is inserted

• Elongation: adding rNTPs to chain – Sigma subunit dissociates after a few bases

are added to the chain

• Termination: dissociation of core polymerase and release of RNA transcript

Phases of transcription

RNA Polymerase

RNA Polymerase

E. coli RNA Polymerase

• Sigma subunit recognizes the transcription start site

• Several different sigma subunits that recognize different promoters

Promoters • Sequences that regulate the efficiency of

transcription initiation• Can be strong or weak • Contain palindromic (e.g. RADAR) consensus

sequences recognized by sigma subunit • TTGACA – sigma70 subunit recognition

domain is always at -35 • TATAAT - Pribnow (or TATA) box always

at -10 for unwinding of helix • Distance between TATAAT and TTGACA

very important for polymerase binding (~17 bp)

Prokaryotic Promoters

•-35 and 10 regions recognized by regions 2 & 4 of σ70 factor•extended -10 recognized by region 3•Region 4 has helix-turn-helix DNA binding motif•1 helix interacts with major groove at -35 & other lies on top of groove interacting with bases

helix interacts with -10 region on nontemplate strand•UP-element recognized by CTD

=

Sigma 70 binding sites

Region 3.2 acts as molecular mimic in abortive initiation; region 2.3 melts DNA

Open complex- note regions of binding by σ70

binds to UP-element

The Transcription Unit

Transcription Occurs in a Bubble

Synthesis Occurs in the 5´ to 3´ Direction

Initiation

Transcription Requires Gyrase and Topoisomerase

Termination• Intrinsic termination - caused by a

palindromic sequence in the DNA template that results in the formation of a hairpin loop that prevents elongation

• Rho-dependent termination - protein physically interacts with RNA transcript preventing elongation. Most gene transcription is terminated this way in prokaryotes

• Antitermination - viral protein allows polymerase to read through the termination sequence making a different protein

Intrinsic Termination

Rho-Dependent Termination

Antitermination

Eukaryotic Transcription

Regulation of Eukaryotic Gene Expression

Transcription Results in an Unprocessed Message

5´ Cap added immediatelyto 5´ sequence, in this case ACATTTG

Poly(A) tail added when sequence (5AATAAA 3) is transcribed

Heterogeneous nuclear RNA (hnRNA) also known as (aka) pre-mRNA or the primary transcript

Translation of mRNA yields Protein

Eukaryotes Have 3 RNA Polymerases

• Pol I synthesizes rRNA in the nucleolus, not inhibited by -amanitin (octapeptide synthesized by a mushroom)

• Pol II synthesizes mRNA and snRNA, inhibited by low concentrations of -amanitin

• Pol III synthesizes 5s rRNA and tRNA, inhibited by high concentrations of -amanitin

RNA Polymerase II

• 12 subunits shaped like a crab claw

• Jaws grip template & clamp locks template at catalytic site for high processivity

• 1 Mg2+ at catalytic site• 8-9 bp of hybrid puts 3´ OH

at catalytic site • 20 bp DNA downstream in

cleft• RNA fits in grooves

• Several channels lead to the active site • 2 DNA channels up and downstream from

transcription bubble make DNA bend about 90°

• Tunnel on opposite side of DNA entry for NTP diffusion and incorporation into RNA

• Rudder protrudes from active site to split RNA-DNA hybrid

• RNA exits from another channel opposite• DNA entry that has a protein flap that may aid

in elongation and termination

A Typical Gene Transcribed by RNAP II

Anatomy of a Gene

Transcription Initiation

Promoters

Overview

• Elements in the promoter can be common to all genes and used constitutively

• Other elements are gene specific: identify particular classes of genes

• These elements exist in different combinations in individual genes

• Housekeeping genes contain elements recognized by general and upstream factors and are transcribed in all cells

Cis-acting Elements Located at a Fixed Distance

from Initiation SiteGeneral elements bound by basal factors for

initiation are called Consensus Sequences• GC box: -110 = GGGCGG, often multiple

copies • CAAT box: -80 = GGCCAATCT • TATA Box: -30 (Goldberg-Hogness Box) =

TATAAAA is pretty nonspecific, fixes initiation site because it is easily denatured

• RNAP II binds at TATA Box

The Promoter Binds General Transcription Factors & RNAP II

In vitro Mutagenesis Shows Critical Sequences for Transcription Initiation

Enhancers• DNA sequences that can modulate

transcription from a distance • Can be upstream, close to start, or

down stream of start site • Aren't always directly involved in

template binding, but are essential to efficient transcription

• Can be negative or positive, but are usually positive

• Position is not fixed - can be upstream, downstream or within an intron

• Can be removed and put back into a different gene and work

• Can be inverted with no effect on activity• Control chromatin structure and rate of

transcription (affect efficiency and stimulate)• Not necessary for transcription but are

necessary for full activation (basal vs. induced expression)

• Are responsible for time and tissue-specific gene expression

• Interact with regulatory proteins & transcription factors

Enhancer Sequences (Response Elements)

Upstream Elements Vary with Gene Function

Modular Nature of Upstream Region for Tissue-Specific Gene

ExpressionNote that many different transcription factors can bind to one gene

It is the set of proteins bound to a gene that determine the level and location of gene expression

Bending DNA Stimulates Transcription

Transcription Factors• Trans-acting factors• Directly facilitate template binding• Essential for transcription initiation because RNAP II

can't bind to promoter and start transcription because eukaryotic chromatin is complexed to protein and promoters are hidden.

• The " factor" for eukaryotes.• Different transcription factors may compete for

promoter/ binding elements, some of which overlap. • Concentration and affinity effect which binds. • Sometimes same binding element binds different

transcriptional factors in a tissue specific manner.• Some are gene specific.

• Basal Factors = TFII s - always associated with Pol II

• True Activators: modular proteins with 2 domains

• DNA-Binding Domain: DNA-Protein Interaction – Distinct structural motifs– DNA-Binding Domains are classified by structural

motif

• Trans-activating Domain: Protein-Protein Interaction

• Can interact with RNAP II or other transcription factors at the promoter and coactivators (hormones, small metabolites)

General (Basal) TFs

Pol II Core Promoter

• Core promoter – minimal set of sequences necessary for accurate initiation

• BRE – TFIIB recognition element• TATA Box• Inr- Initiator• DPE – Downstream element• Typical promoter usually include only 2 or 3

of any of these• Upstream lie regulatory elements

Stages of Initiation

Commitment• TFII D complex binds to TATA box via TATA

Box Binding Protein (TBP) and TAF's (TATA Associated Factors) ~20 bases involved

• TFII D complex contacts DNA - changes conformation to facilitate binding of TFII B and A

• RNAP II complexed to TFII F binds next • TFII E • TFII H (helicase & kinase activity)• NTPs enter • TFII J (?)

Assembling the Basal Complex

Initiation

TAFs Interact with TFIID (& TBP)

TBP-DNA

TFIIB-TBP-DNA

Mediator Complexes• Multiprotein complexes associated with RNAP II

• Do not bind to DNA

• Act like control panels for RNAP II

• Mediate interactions with TFs

• Often required for the function of TFs

• Integrate all of the positive and negative regulatory signals for RNAP II and "determine" how much message should be made.

• Probably interact with the C-terminal domain (CTD) of the largest RNAP II subunit

Mediator Complexes Are Composed of Many

Coactivators

Coactivators Interact with TFs, But Do Not Bind to DNA

Mediators May Stabilize Pre-Initiation Complex After Chromatin

Remodeling

• Mediators associate with the CTD tail

• Interact with DNA-bound activators

Multiple Pathways Affect Transcriptional Activation

Promoter Escape

• Pol II moves away from the promoter• Synthesizes 10-15 nucleotides• Dissociates from general initiation factors • Cannot occur unless CTD is

hyperphosphorylated by TFII H

Promoter Escape Requires CTD Phosphorylation by TFIIH

Other Proteins Associated with Promoter Escape & Elongation

Elongation

Phosphorylation of Ser 2 recruits splicing factorsPhosphorylation of Ser 5 recruits capping factorsOther factors include TFIIS, P-TEFb, TAT-SF1

Abortive Transcriptionand Proofreading

• Transcripts smaller than about 9 nucleotides are aborted

• TFII F acts to decrease abortive transcription (by increasing rate of polymerization?)

• TFIIS contributes to Pol II’s proofreading by stimulating its inherent RNase activity

Arrested Transcription• Transcription can also be arrested at promoter

escape, potentially by TFII F binding to promoter ahead of transcriptional start site

• Can be suppressed by TFII E & TFII H-XPB DNA helicase (ATP-dependent) activity which act to disrupt TFII F's interaction with the promoter

• TFII H also recognizes damaged template DNA and recruits proteins for DNA excision-repair

• Mutations in TFII H can result in diseases with sensitivity to light and increased risk of cancer such as xeroderma pigmentosum, trichthiodystrophy, or Cockayne syndrome (depending on mutation severity)

EFs Can Reactivate Arrested RNAP II

• The SII family of EFs reactivate stalled Pol II by cleaving the transcript upstream of the 3´-OH of the last nucleotide making a new 3´ end so that RNAP II can add new nucleotides

EFs Can Prevent RNAP II Arrest

• P-TEFb is a cyclin-dependent kinase that phosphorylates CTD to prevent elongation arrest

• DSIF and NELF are negative regulators of elongation – Both interact with Pol II in its

hypophosphorylated form– DSIF/NELF blockade is removed by P-TEFb

phosphorylation of CTD

RNAP II Pausing Is the Rate-limiting Step in Elongation

• EFs can prevent pausing

• TFII F, ELL, Elongin & CSB suppress pausing by decreasing the time RNAP II spends in an inactive conformation increasing rate the of transcription

EFs Modify Chromatin Structure

• HMG14, FACT & Elongator modify and destabilize nucleosomes clearing the path for RNAP II movement

• Elongator and SWI/SNF remodel chromatin

Elongation

Transcription Visualized

Prokaryotic Eukaryotic

RNA processing is coupled to elongation

• RNAP II CTD interacts with RNA processing proteins to process the transcript as it comes through the flap at the end of tunnel

• 7-methyl guanine cap, splicing and polyadenylation are coupled to elongation

Capping the 5´ end of the transcript

• The 7-methyl guanosine cap is added to the 5´-PO4 before the transcript is 30 nucleotides long

• May be used to attenuate mRNA output

• Unique 5-5 bond is added shortly after transcription initiation via 3 reactions

Capping reactions

1. Phosphatase removes 5 phosphate

2. Guanylyl transferase catalyzes a condensation between the 5 triphosphate and GTP

3. Guanine-7-methyltransferase transfers the methyl group

4. Ser 5 of CTD dephosphorylated and capping machinery leaves

• All eukaryotes possess a methyl on N7 of the terminal guanine

• Higher eukaryotes often add a second methyl group to the penultimate base at the 2-O position (2-O-methyltransferase)

3 Polyadenylation• Ser 2 must be phosphorylated for

poly(A) factor recruitment• Length of poly(A) tail determined

by proteins bound to poly(A) sequence

• AAUAA signals the addition of the poly(A) tail

• Poly(A) polymerase adds ~200 A residues to the free 3-OH of the transcript

• Poly(A) tail leads to cleavage ~10-35 upstream of signal

• Cleavage polyadenylation stimulatory factor (CPSF) recognizes the polyadenylation sequence (AAUAAA)– associates with TFIID first, then

jumps on to CTD after initiation • Cleavage stimulatory factor

(CstF) also interacts with CTD– necessary for elongation

Poly(A) Tail Confers Stability to mRNA

• Poly(A) tail is associated with the poly(A)-binding protein (PABP)

• Poly(A) tail + PABP thought to confer stability to many mRNA transcripts and is involved in translation initiation

RNA Splicing

• As pointed out earlier, the concept of a gene having protein-coding sequences interrupted by non-coding sequences was not recognized until the late 1970’s

• Work in Phil Sharp’s lab at MIT by his post-doc Sarah Flint demonstrated that eukaryotic gene structure differed from prokaryotic gene structure

• Walter Gilbert named these gene regions:– Exon = expressed sequences– Intron = intervening sequences

Splicing Visualized

Transcription initiated here

Introns loop out as they are excised

Splicing Mechanisms

Introns Are Classified by Their Splicing Mechanism

Splicing involves two transesterifications

Trans-Splicing joins exons from two different RNAs

Self-excising group I & 2 introns

2 nucleophilic transesterification reactions

3-OH guanosine on right sideof intron is transferred to nucleotide at 5 end of intron

Guanosine acts as cofactor

"New" 3-OH on left side of intron and phosphate group on 3 end of right side of intron interact leaving phosphate for ligation of exons 1 and 2

Spliceosome

• snRNA small nuclear RNAs • snRNPs small ribonucleoproteins (snurps)

– rich in uridine – only in the nucleus – designated U1, U2, etc.

• Serine-Arginine (SR) proteins act as bridging factors– N- terminal RNA recognition motifs for binding

hnRNA– C-terminal arg-ser (RS) rich sequences for

protein-protein interactions with RS domains in snurps

hnRNPs Involved in Splicing Reactions

Nuclear Splicing

1. U1 binds to exon 1-intron (5 splice site) binding site

2. U2, U4, U5 & U6 bind, splicing begins (2 trans-

esterification reactions) 3. 2-OH from branchpoint(internal adenine residue) of intron attacks 5 splice

site & cuts polymer

4. Free OH created at the end of exon 1 attacks the intron-exon 2

junction

5. Introns excised

6. Exons ligated

Assembling the Splicesome

Splicing and errors

• Errors are decreased by coupling transcription and splicing – see 3′ site as transcribed so no competition from other sites

• Errors decreased by exonic splicing enhancers – ser arg rich sites that are bound by the essential SR proteins that recruit snurps to splice sites

• SR proteins also necessary for alternative splicing

Putting It All Together

Alternative Splicing Regulates Gene Expression

Alternative splicing

Alternative splicing results in families of proteins (splicing isoforms)

Types of Alternative Splicing

(a) Alternative 5´ splice site(b) Alternative 3´ splice site(c) Skipping thevariable alternative splice exon(d) Mutual exclusionof exons(e) Gender-specific splicing

Alternative Poly(A) site

Found in prostate cancer

Preprotachykinin (PPT) Gene

P

P

Splicing is regulated

Combinatorial Control

Sex lethal binding results in stop codon being spliced out

Functional transformer binding causes doublesex to be spliced in a female-specific fashion

Stop codon remains

Transformer not functional

Male-specific doublesex

Exon shuffling

RNA Editing

Changes the sequence of the RNA after transcription, but before

translation

Insertion/Deletion Editing

• Nucleotide addtion or subtraction directed by guide RNA (gRNA) templates

• Add poly(U) to form initiation codon and set reading frame

• gRNA template complementary to edited region of final RNA transcript

• gRNA base pairs with pre-RNA and directs editing complex to make appropriate changes to RNA transcript

gRNA Directs T. brucei RNA Editing

Substitution editing

• Nucleotides are altered by substituting one for another

• Prevalent in mitochondria and chloroplasts • Apolipoprotein B (apo B) exists in long and short

forms• Intestine: protein complex binds to "mooring"

sequence downstream of editing site • C to U substitution: CAA = glutamine; UAA =

stop (short form)

Apo-B Gene Is Modified by RNA Editing

Transcription-Induced Z-DNA, dsRNA & RNA Editing

• Z-DNA stabilized by negative supercoiling induced by RNAP II

• dsRNA editing substrate forms by 3´ intron folding back on exon to be edited

• Adenosine Deaminase Acting on RNA (ADAR) 1 Binds to dsRNA and Z-DNA

• It is proposed that binding to Z-DNA allosterically activates ADAR1

• ADAR1 deaminates adenosine to inosine • I read as G during translation resulting in glutamine

(CAG) to arginine (CGG) substitution • Occurs in Glutamine Receptor-B and Serotonin-2C

receptor

ADAR1 Mechanism

Antisense (RNAi, siRNA and miRNA) Regulation of Translation

• All use a large dsRNA that activates an enzyme called dicer

• Dicer digests large transcript into short pieces (21-23 nt) that recruit the RISC complex of proteins

• The RNAi (siRNA or miRNA) then bind to the target resulting in translational arrest, digestion of newly-formed dsRNA or promoter silencing via chromatin modification

Kosik Nature Reviews Neuroscience 7, 911–920 (December 2006) | doi:10.1038/nrn2037

RNAi/miRNA Mechanism

C. elegans makes lin-4 antisense to regulate lin-14 expression

Now consider lin-4 antisense

an miRNA

Have Many Antisense "Drugs" in Clinical Trials

• Vitravene is an antisense “drug” that targets cytomeglavirus in AIDS patients with cytomeglavirus-induced retinitis

• ICAM-1 (inflammatory cell adhesion molecule-1) antisense causes remission in 50% of Crohn's disease patients in clinical trials

Nuclear transport

Regulating mRNA Stability

• Information in 5 and 3 untranslated sequences important

• Stability sequences increase half life (t1/2 )

• Instability sequences decrease t1/2

– AUUUA rich sequences of ~50 bases (ARE) is bound by an ARE-binding protein

– Causes mRNA to be deadenylated & lose PABP

– Digested by poly(A) ribonuclease

– Endonucleases digest RNA

Decreased mRNA stability reduces amount of protein made

and tubulin levels demonstrate translational control

• Add colchicine microtubules dissociate and subunit concentrations increase causing

tubulin synthesis to drop

• Add vinblastine microtubules dissociate and are precipitated and subunit synthesis increases

• Difference probably caused by binding of free subunits to specific AA sequence encoded by 5 nucleotides

• Protein-protein interaction activates an RNase that digests template

Altering mRNA Stability Allows for Translational Control

Prokaryotic Regulation

Operons

Operons

• Units of transcription used to regulate gene expression in prokaryotes

• Genes are grouped together in clusters for response to environmental conditions

• Expression of cluster is regulated from one site • Inducible - are turned on in response to the

presence of the substrate (the inducer) for a necessary enzyme

• Repressible - presence of a specific molecule (the repressor) that inhibits gene expression

Activation of gene expression

Recruitment of polymerase Allosteric activation

Cooperative binding and DNA bending activate gene expession

Negative ControlGene is expressed unless it is turned off by some regulatory molecule

Positive ControlGene only expressed if a regulatory molecule stimulates RNA synthesis

The lac Operon

Prokaryotic genes do not have introns and exons makepolycistronic mRNA - continuous transcripts that are

composed of many genes.

Expression of lac genes

Control region

Negative Control

Repressor binds to operator but activator interacts with CTD tail

Inducer Changes Repressor Conformation

Operators

Lac Operon Has 3 Operators

All 3 operators must be bound for maximal repression. Repressor binding to 2 operators causes a DNA conformational change DNA bends away from the repressor forming a repression loop, preventing RNA polymerase access to the promoter

Operator Mutations Are Constitutive

lacI Mutations Are Constitutive

Repressor Mutants Are Super-repressed

Catabolite Repression

trp Operon

• Leader is composed of 162 nucleotides that contains another regulatory region, the attenuator

• Tryptophan (Trp) is a co-repressor

• When Trp binds to the normally inactive repressor protein, the repressor can bind to the operator and inhibit expression of the operon

trp operon is repressible

Transcription Occurs in the Absence of Tryptophan

Repressor Binds in the Presence of Tryptophan

Attenuation• Attenuation only occurs in the presence

of tryptophan • When the operon is repressed,

transcription of the leader sequence is initiated but not completed

• Transcription is attenuated 140 nucleotides into the leader sequence

• Hairpin loop formed by the RNA encoded by the DNA in the attenuator region

• Loop is followed by a polyU tract

Leader Sequence

• Leader encodes 2 triplets (UGG) that encode trp.

• Leader also contains a translation initiation codon (AUG)

Attenuation Is Dependent Upon Leader Sequence

trp Leader Sequence

Tryptophan Available

• If trp is plentiful, charged trp-tRNA is present and translation occurs

• Leader is made and the hairpin loop is formed

Tryptophan Present

Low Tryptophan Concentrations

• Charged trp-tRNA is not available and translation of the leader can not occur because the ribosome stalls

• Ribosome stalling affects secondary structure of transcript no hairpin loop is formed

• Transcription of the structural genes proceeds

Low Tryptophan

Translation

tRNA

• Transcribed as one large primary transcript that is cleaved into smaller 4s tRNA molecules (70-90 nucleotides)

• Have extensive post-trancriptional modifications

• Have secondary and tertiary structure

• 32 different tRNAs due to wobble position

• Nomenclature: Phenylalanyl tRNA = tRNAphe where phe is the cognate AA

tRNA contains rare nucleotides

• Rare nucleotides, i.e., pseudouridine, inosine, etc.

• Some are observed in all tRNAs (dihyrouridine in D loop, pseudouridine () in TGC loop of acceptor stem, etc.), while others are specific for a particular tRNA or group of tRNAs.

tRNA exhibits atypical basepairing

tRNAs exhibit secondary and tertiary structure that results in the formation of

loops and stems

Cloverleaf Model (Holley, 1965) • Anticodon loop - binds to codon of mRNA • Acceptor stem - necessary for tRNA charging by tRNA

synthetase. Places the terminus of the tRNA close to the active site of the enzyme

• Amino acid (AA) binding site - contains the sequence 5 CCA 3 • Variable loop - also necessary for synthetase recognition

Aminoacyl tRNA Synthetases

• Very specific for AA and isoaccepting tRNA (cognate tRNA)

• Cognate tRNA: multiple tRNAs that represent the same AA

• Recognizing only one AA and tRNA is essential for the FIDELITY of the system b/c ribosome blindly accepts any charged tRNA with proper codon-anticodon interaction

• Acceptor stem has a discriminator base at 3 acceptor end that is especially important for recognition specificity of a tRNA from 1 synthetase to another

• Anticodon loop also contributes to discrimination

Synthetase Structure

Amino Acids

tRNA Charging

1. Synthetase + AA +ATP aminoacyl~adenylic acid-synthetase complex + PPi

2. Synthetase-aminoacyl~adenylic acid complex + tRNA synthetase + charged tRNA + AMP

Chemistry of Charging

• Step one is an adenylylation: AA reacts with ATP, AMP transferred, PPi released

• This step results in a high-energy ester bond joining the AA and AMP.

• Breaking of this bond during peptidyl transferase reaction provides energy for formation of the peptide bond.

• Step 2 is tRNA charging where AA reacts with tRNA

Recognition of correct tRNA

Biological polymerization of AA into polypeptide chains

Ribosome structure

• Composed of catalytic rRNA and structural proteins

• Large and small subunits • rDNA mildly repetitive • Exists in clusters of tandem repeats (repeating

sequences over and over) • All ribosomal RNA is transcribed as one large

primary transcript followed by cleavage into smaller functional transcripts

Prokaryotic Ribosome•Small subunit has decoding center•Large subunit has peptidyl transferase center

Prokaryotic vs. Eukaryotic Ribosome

Electron Micrographs of the Ribosome

Ribosome Active Sites

Mechanisms and Process

Transcription and translation are coupled in prokaryotes

OverviewPolyribosomes

Important Sequences for Initiation and Setting the Reading Frame

• Ribosome binding site (RBS) aka Shine-Delgarno sequence (-10): 5AGGAGG 3

• Complementary to 16s rRNA sequence: 3UCCUCC 5

• Start codon: 5AUG 3 (sometimes GUG or UUG)

– Encodes initiator tRNA: fMet-tRNAi

fMet = N-formyl methionine (different tRNA used for internal AUG)

– Deformylase removes formyl group if Met is 1st AA

– Aminopeptidase removes Met if not 1st AA

Ribosome Has 2 Sites for Binding Charged tRNA

fMet-tRNAifMet

Has 3 GC pairs in stem before anticodonloop that is necessary for entrance into P site

ONLY fMet-tRNA Can Enter the P Site

Binding Sets the Reading Frame!!!

fMet Removed During Synthesis

Initiation

Initiation Factors

• Initiation factors (IF) absolutely required

• Never observed in 70s ribosomal structure

• All IFs released and GTP hydrolyzed so that 50s can bind

• IF1- stabilizes initiation complex

IF2

• Essential for entry and binding of fmet-tRNA into P site

• Binds GTP • Ribosome-

dependent GTPase activity for formation of 70s ribosome

IF3

• Stabilizes free 30s subunits

• Prevents association of 50s

• Dissociates ribosome into subunits at termination

1. IF3 occupies E site 2. IF1 binds to A site and IF2 binds to it leaving only P site open3. fMet-tRNAi

fMet binding is facilitated by interactions with IF2-bound GTP

4. When start codon and initiator base-pair, small subunit changes conformation and releases IF3

5. Large subunit (50S) binds and stimulates IF2 GTPase activity, GTP hydrolyzed

6. IF2-GDP and IF1 released7. 70S ribosome formed allowing a charged tRNA to enter A site

Antibiotics Inhibit Translation

Eukaryotic Translation• Translation initiation does not involve a Shine-

Delgarno sequence • Kozak sequence (5 ACCAUGG 3) surrounds

initiator codon• Ribosomes enter at the 5 cap and advance to

the first AUG via small subunit linear scanning • Start recognized by anticodon of initiator tRNA

which is why initiator is bound to small subunit prior to ribosome assembly

• Control is usually exerted at the rate-limiting initiation step

43s pre-initiation complex plus eIF4F/B bound mRNA form 48s initiation

complex40s subunit binds to eIF1A + eIF3.Next, eIF5B-GTP + eIF2GTPMet-tRNAi

Met associate with small subunit and position initiator in P site

• The cap-binding-protein complex = eIF4F finds the cap, acts as an RNA helicase to unwind 5 mRNA secondary structure

• eIF4F has 3 subunits: – A has RNA-dependent ATPase

activity– E binds the cap – G is a docking site for the

initiation complex - acts as the central adapter for the binding of regulation and initiation factors

• eIF4B activates helicase of eIF4F• eIF4F/B recruits 43s pre-initiation

complex to mRNA via eIF3 = 48s initiation complex

43s

Scanning to find the initiator

• Scanning is ATP-dependent and requires eIF4F to drive scan via its helicase activity

• Find AUG & base-pair • eIF2, eIF3 and 4B

released • Large subunit (60s)

binds, stimulates eIF5B hydrolysis of bound GTP

• 5B-GDP & 1A released • Form 80s ribosome

Translation Requires Template Circularization

• eIF4F G subunit interacts directly with the poly(A) tail binding protein (PABP), and mRNA

• When all of the initiation factors are bound, the mRNA template is circularized

• Template circularization is thought to facilitate re-initiate translation

Translation Is Tightly Regulated

Affinity of eIF4E for cap increased with phosphorylation

MAP kinaseinteracting protein 1

PKC

Preiss and Hentze, 1999Current Opinion in Genetics & Development

eIF4E Binding Proteins Compete for Binding with

eIF4G

Sonenberg & Gingras, 1998

eIF4E, A and G = eIF4F complex

Control at initiation by numerous signal transduction

pathways

Many signal transduction pathways converge on eIF4E and phosphorylate it as well as other IF factors & ribosomal proteins

Sonenberg & Gingras, 1998

eIF,

Devers, 1999

Elongation Factors • EF-Tu

– Mediates entry of incoming aa-tRNA into A site – GTP is hydrolyzed after codon-anticodon recognition– Leaves after aa-tRNA is in A site – Does NOT recognize fmet-tRNA, only IF2 does – Function inhibited by the antibiotic kirromycin causing EF-Tu to

remain bound to the ribosome– EF-TuGDP inactive, can't bind aa-tRNA – EF-TuGTP active, can bind aa-tRNA

• EF-Ts – a GTPase exchange factor– Regenerates EF-TuGTP by displacing GDP from EF-Tu

• EF-G – Stimulates translocation (movement of ribosome 3 nucleotides

downstream) – Release requires GTP hydrolysis– EF-GGTP regenerated from EF-GGDP b/c GTP has higher affinity

for EF-G than GDP does

Elongation and the ribosome

• The ribosome is a ribozyme – the 23S rRNA (large subunit) catalyzes peptide bond formation

• Ribosome very accurate – uses 3 mechanisms in addition to codon-anticodon interactions to select against incorrect codon-anticodon pairings:

1. Two adenine residues in 16s rRNA form tight interaction with minor groove of correct base pair. Don’t recognize non-Watson-Crick base pairs b/c form a minor groove they don’t recognize, significantly reducing affinity for mismatches.

2. Proofreading - One mismatch dramatically reduces GTPase activity of EF-Tu. Mechanism very similar to #1.

3. Accomodation – a form of proofreading that occurs after EF-Tu is released. tRNA is moved closer to peptidyl transferase center by rotation. Incorrectly paired tRNA will usually dissociate here.

Steps in Elongation1. EF-TuGTP binds to tRNA 3 end masking AA2. EF-TuGTPaa-tRNA binds to A site3. Correct codon-anticodon match is made4. Factor binding center activates Ef-Tu GTPase, GTP hydrolyzed,

EF-TuGDP is released5. Peptidyl transferase hydrolyzes bond between tRNA and AA

yielding energy for peptide bond formation 6. Peptide bond formed between AAs in P site and A site and

peptide transferred to A site7. tRNA in P site is deacetylated and uncharged tRNA moves to E

site 8. EF-GGTP binds to A site on large subunit, contacts factor

binding center9. GTP hydrolyzed, ribosome translocation (ribosome moves 3

nucleotides 3 on mRNA) A site empty, EF-GGDP is released

10.Peptide chain emerges from tunnel in large subunit (after 30 aa are visible)

OverviewEF-Tu carries

aa-tRNA to A site

Peptide Bond Formation

Translocation

Translocation

Termination Factors• Recognize stop codon, catalyze dissociation of ribosome

• 2 Class I Releasing Factors in prokaryotes (RF1 and 2) and 1 in eukaryotes (eRF1)

• Class I RFs mimic tRNA and result in hydrolysis of the peptide chain from the tRNA in the P site – Have a peptide anticodon that recognizes and interacts

with the stop codon• Class II RF stimulate release of Class I RFs and are regulated by GTP – 1 Class II RF in both prokaryotes and eukaryotes = RF3 &

eRF3, respectively– Has high affinity for GDP NOT GTP

• Ribosome recycling factor (RRF) mimicks a tRNA in the A site and recruits EF-G– Cooperates with IF3 and EF-G to remove tRNAs from E and

P sites and release mRNA

Steps 1.RF1 (UAA or UAG) or RF2 (UAA

or UGA) see stop codon 2.No aa-tRNA for stop codons so

nothing in A site 3.RF1/2 mimic tRNA, activate

ribosome to cleave peptide 4.RF3-GDP binds to ribosome,

exchanges GDP for GTP causing release of RF1/2

5.RF3-GTP interacts with the factor binding center of the ribosome causing GTP hydrolysis and RF3 release from the ribosome

6.RRF & EF-G cause tRNA to leave P & E sites

7. IF3 binds to small subunit causing dissociation of ribosome

What happens if a ribosome stalls?

• A chimeric molecule called a tmRNA mimics tRNA and mRNA

• For example, SsrA (charged with an alanine) can bind to EF-Tu-GDP, enter the A site and cause translocation and release of mRNA

• In addition, a part of SsrA enters the mRNA channel of the ribosome and extends the ORF by 10 codons and a stop codon

• This results in a protein with 10 extra AA that tag the protein as incomplete, causing cellular proteases to digest it

What does the cell do if there is an early stop codon?

• Normally, exon junction complexes (from splicing) are removed during translation

• If a premature stop codon (a nonsense mutation) is encountered then the complexes still exist downstream from the mutation b/c translation stops before whole protein is made

• This activates the nonsense mediated decay process where the remaining exon junction complexes recruit Upf proteins to the ribosome. Upf proteins activate the decapping enzyme that removes the 5 cap resulting in degradation of the mutated mRNA by 5 3 exonuclease.

What happens if there isn’t a stop codon?

• Nonstop mediated decay rescues the ribosome by recognizing that the ribosome has translated the poly-A+ tail and stalled the ribosome

• The stalled ribosome is bound by the exosome that includes Ski7, a protein related to eRF3, that causes ribosome dissociation and a 3 5 exonuclease that digests the mutated message

• The poly-lysine tag on the carboxy terminus of the protein activates cellular proteases and the mutant protein is digested

A Translation Movie

Translation Animation Web Addresses

http://www.geocities.com/CapeCanaveral/Lab/5451/transgif.htm

http://tidepool.st.usm.edu/crswr/protsynthmov.html

http://www.bio.cmu.edu/Courses/BiochemMols/ribosome/70S.htm

http://www.ncc.gmu.edu/dna/ANIMPROT.htm