Prokaryotic Transcription

Transcription
DNA-dependent RNA synthesis
RNA polymerase doesnt 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
helix interacts with -10
region on nontemplate strand
UP-element recognized by
CTD

-35 and 10 regions recognized by regions

2 & 4 of 70 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

Sigma 70 binding sites

Region 3.2 acts as molecular mimic in

abortive initiation; region 2.3 melts DNA

binds to
UP-element

Open complex- note regions

of binding by 70

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

5 Cap added immediately

to 5 sequence, in this case
ACATTTG

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

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 Site
General 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
Expression
Note 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
DNABindingDomainsareclassifiedbystructural
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)

TFIIB-TBP-DNA
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 PreInitiation 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 factors

Phosphorylation of Ser 5 recruits capping factors
Other 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 IIs 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 Ratelimiting 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.
2.
3.
4.

Phosphatase removes 5
phosphate
Guanylyl transferase catalyzes a
condensation between the 5
triphosphate and GTP
Guanine-7-methyltransferase
transfers the methyl group
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 1970s
Work in Phil Sharps 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 side
of 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 proteinprotein 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 transesterification reactions)
3. 2-OH from branchpoint
(internal adenine residue)
of intron attacks 5 splice
site & cuts polymer
5. Introns excised
6. Exons ligated

4. Free OH created at the end of

exon 1 attacks the intron-exon 2
junction

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

Alternative Poly(A) site

Found in prostate cancer

(a) Alternative 5
splice site
(b) Alternative 3
splice site
(c) Skipping the
variable alternative
splice exon
(d) Mutual
exclusion
of exons
(e) Gender-specific
splicing

Preprotachykinin (PPT)
Gene

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 newlyformed dsRNA or
promoter silencing via
chromatin modification

RNAi/miRNA Mechanism

Kosik Nature Reviews Neuroscience 7, 911920 (December 2006) | doi:10.1038/nrn2037

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 Control
Gene is expressed unless it is turned off by some regulatory molecule

Positive Control

Gene only expressed if a regulatory

molecule stimulates RNA synthesis

The lac Operon

Prokaryotic genes do not have introns and exons make

polycistronic 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 posttrancriptional 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 acidsynthetase 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

Overview

Polyribosomes

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: fMettRNAifMet = 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-tRNAi

fMet

Has 3 GC pairs in stem before anticodon

loop 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
Ribosomedependent
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 open
3. fMet-tRNAifMet binding is facilitated by interactions with IF2bound 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 released
7. 70S ribosome formed allowing a charged tRNA to enter A site

Antibiotics Inhibit
Translation

Eukaryotic Translation
Translation initiation does not involve a ShineDelgarno 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
complex
40s subunit binds to eIF1A + eIF3.
43s

Next, eIF5B-GTP + eIF2GTPMettRNAiMet 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

Scanning to find the

initiator
Scanning is ATPdependent 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 reinitiate translation

Translation Is Tightly
Regulated
MAP kinase
interacting
protein 1

PKC

Preiss and Hentze, 1999

Current Opinion in Genetics
& Development

Affinity of eIF4E for cap increased with phosphorylation

eIF4E Binding Proteins

Compete for Binding with
eIF4G
eIF4E, A and G = eIF4F complex

Sonenberg & Gingras, 1998

Control at initiation by
numerous signal transduction
pathways

Devers, 1999
eIF,
Sonenberg & Gingras, 1998

Many signal transduction pathways converge on eIF4E and phosphorylate it as

well as other IF factors & ribosomal proteins

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

1.

2.
3.

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:
Two adenine residues in 16s rRNA form tight interaction
with minor groove of correct base pair. Dont recognize
non-Watson-Crick base pairs b/c form a minor groove
they dont recognize, significantly reducing affinity for
mismatches.
Proofreading - One mismatch dramatically reduces
GTPase activity of EF-Tu. Mechanism very similar to
#1.
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 Elongation
1.
2.
3.
4.

EF-TuGTP binds to tRNA 3 end masking AA

EF-TuGTPaa-tRNA binds to A site
Correct codon-anticodon match is made
Factor binding center activates Ef-Tu GTPase, GTP hydrolyzed,
EF-TuGDP is released
5. 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 site
7. 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 center
9. 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)

Overview

EF-Tu carries
aa-tRNA to A site

Peptide Bond Formation

Translocation

Translocatio
n

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 EFTu-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 isnt 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.bio.cmu.edu/Courses/BiochemMols/ribosome/70S.htm
http://www.geocities.com/CapeCanaveral/Lab/5451/transgif.htm
http://www.ncc.gmu.edu/dna/ANIMPROT.htm
http://tidepool.st.usm.edu/crswr/protsynthmov.html