transcription and translation
TRANSCRIPT
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