Transcript PPT

Translational Recoding
David Bedwell
Post-Transcriptional Regulatory Mechanisms
Advanced Course
MIC743
Feb 22, 2011
Myths in Modern Molecular Biology
 The “universal genetic code” is universal.
 The genetic code is unambiguous.
 All DNA (and RNA) genomes encode the information to make proteins
with only 20 amino acids.
 The “central dogma of molecular biology” (DNA  RNA  protein)
describes the only flow of biological information.
 Eukaryotic translation initiation only occurs in a cap-dependent
manner (à la Kozak).
Recoding mechanisms frequently represent exceptions to
established dogma and highlight functional features of
underlying mechanisms of gene expression.
Lecture Overview
 Background: examples of recoding that will allow us to
understand more translation.
o Ribosomal frameshifting
o Ribosome hopping
o Incorporation of unusual amino acids at stop codons
o Stop codon readthrough
 Pharmacological suppression
 Suppressor tRNAs
o Switching mRNA templates during translation
 Clinical implications:
o Induced recoding may help treat many genetic diseases
o Prevention of recoding may help treat some infectious
diseases.
Comparison of Eukaryotic and Prokaryotic
Elongation Factors
Prokaryotes
Eukaryotes
EF-Tu (GTPase)
eEF1A (GTPase)
EF-Ts (GEF)
eEF1B (GEF)
EF-G (GTPase)
eEF2 (GTPase)
Translation Elongation
e)
50S
EF2:GDP
d)
50S
AA-tRNA
AA-tRNA:EF1A:GTP
30S
Translocation
& GTP
hydrolysis
EF1A:GTP
a)
30S
EF1B
50S
GTP
tRNA
selection
30S
EF1A:EF1B
Hybrid
state
EF2:GTP
c)
50S
GTP hydrolysis
& proofreading
Peptide
bond
formation
b)
50S
GDP
EF1A:GDP
EF1B
30S
30S
Merrick & Nyborg, The Protein Synthesis Elongation Cycle, In
Translational Control of Gene Expression (2000), CSHL Press, NY
tRNA Selection During Elongation
Ramakrishnan, Cell 108: 557-572 (2002)
Recoding can occur here
Comparison of Eukaryotic and Prokaryotic
Termination Factors
Prokaryotes
Eukaryotes
RF1 = UAA, UAG
eRF1 = UAA, UAG, UGA
RF2 = UAA, UGA
-
RF3 = GTPase
eRF3 = GTPase
Prokaryotic Translation Termination
Zavialov et al., Cell 107: 115-124 (2001)
Eukaryotic Translation Termination
Alkalaeva et al., Cell 125: 1125-1136 (2006)
A Closer Look at the Fidelity of tRNA Selection:
SSU Helix 44 is a Key Determinant of Fidelity
Helix 44
“Decoding Site”
Ramakrishnan, Cell 108: 557-572 (2002)
Yusupov et al., Science 292: 883-896 (2002)
Critical Nature of A1492 and A1493 in Helix 44
of 16S rRNA in Translational Fidelity
(Helix 34)
(Helix 18)
(Helix 44)
Ogle et al. (2002) Science 292:897-902.
(Helix 44)
Aminoglycosides Bind Helix 44 and Reduce
Ribosomal Fidelity During Translation
Unbound
- tRNA
+ tRNA
Ogle et al, Science 292:897-902 (2001)
Paromomycin bound
A Competition Occurs When Any Codon
Enters the Ribosomal A Site
Example: Stop Codon Recognition)
GTP
eRF1 eRF3
m7GpppG
AUG
CAG UAA
P
Competitions:
• Stop codon vs. near-cognate codons
• Sense codon vs. near-cognate codons
A
UAA
(Normal Freq ~99.9%)
AAAAAAAAA
eEF1A
Nearcognate
AA-tRNA
Termination
GTP
Readthrough
(Normal Freq ~0.1%)
Various Conditions can Shift This Balance to
Increase the Frequency of Mis-Incorporation
GTP
Termination
eRF1 eRF3
m7GpppG
AUG
CAG UAA
P
A
A key recoding Inducer is pausing caused by:
• Hungry codons
• mRNA structures
Recoding enhanced by:
• Slippery Sites in mRNA
• Dedicated factors
UAA
AAAAAAAAA
eEF1A
Nearcognate
AA-tRNA
(90-95%)
GTP
Readthrough
(5-10%)
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Feedback Control of RF2 Expression in Bacteria
 RF-2 recognizes UAA and UGA, while RF-1 recognizes UAA
and UAG stop codons.
 The RF-2 ORF contains an in-frame UGA stop codon and a
modest Shine-Dalgarno (SD) sequence 5 nucleotides upstream
of the frameshift site (5´-AGGGGGU-3´).
 When the RF-2 level is low, the ribosome pauses when a UGA
codon is located in the A site. tRNAleu in the P site then slips
from the CUU codon to the UUU codon.
 Frameshifting is enhanced by the presence of the SD-like
element (thought to re-establish the ribosome in the new
reading frame).
 In this way, more RF-2 is made when there is not enough to
rapidly terminate translation at the UGA stop codon.
+1 Frameshifting Required for E. coli RF-2 Synthesis
*
*
* * *
E. coli
S30
RRL
Donley & Tate, Proc Biol Sci 244: 207-210 (1991)
Namy et al., Mol Cell 13: 157-1698 (2004)
Cellular Polyamine Levels Control Antizyme 1
Synthesis via a +1 Frameshifting Mechanism
 Polyamines like spermine and spermidine are found in both
prokaryotes and eukaryotes, where they stabilize membranes,
ribosomes, DNA, viruses, etc.
 Cellular polyamine levels are regulated by antizyme 1 in eukaryotes.
 High polyamine levels stimulate the synthesis of antizyme 1.
 Antizyme 1 then binds to ornithine decarboxylase (ODC) and triggers
its degradation by the 26S proteosome (in an unusual ubiquitinindependent manner).
 Since ODC catalyzes the 1st step in polyamine synthesis, its
degradation leads to reduced polyamine synthesis.
 Reduced polyamine levels reduce antizyme 1 expression.
 Antizyme expression controlled by +1 frameshifting mechanism
induced by high polyamine levels.
 Required elements include polyamines, a “shifty stop” slippery
sequence (5´-UCC UGA U-3´) at the frameshift site, and a pseudoknot
just 3´ of the slippery sequence that induces a ribosomal pause.
+1 Frameshifting in Antizyme Synthesis
Pseudoknot
Structure
poorly defined 5´
stimulatory sequence
Namy et al., Mol Cell 13: 157-1698 (2004)
“shifty stop”
slippery sequence
(5´-UCC UGA-3´)
+1 Frameshifting in the yeast EST3 gene
 EST3 encodes a subunit of telomerase with an internal programmed +1
frameshift site between ORF1 (93 AAs) and ORF 2 (92 AAs) in S.
cerevisiae and also many other yeast species.
 The frameshift site has the slippery sequence 5´-CUU AGU U-3´.
 AGU is encoded by a low abundance tRNA (sometimes referred to as a
“hungry codon”), which frequently induces a ribosomal pause.
 During pausing, the tRNAleu in the P site can undergo +1 slippage to
the overlapping UUA codon.
 May be other required elements, but not known yet.
EST3 +1 frameshifting is conserved in
many yeast species
Conservation of this slippery site among many related yeast species over millions of years of
evolution suggests frameshifting may play some important role in telomere maintenance.
Namy et al., Mol Cell 13: 157-1698 (2004)
-1 Frameshifting is Common in Retroviruses
(Including HIV) and Other Viruses
Model of Beet Western Yellow Virus (BWYV) -1 frameshift. Bases in red are conserved
in all known luteoviruses. Frameshifting requires:
•
•
7 nucleotide slippery site
downstream pseudoknot
Alam et al., Proc Natl Acad Sci USA 96: 14177-14179 (1999)
Retroviral -1 Frameshifting
 Retroviral -1 frameshifting between the Gag and Pol reading
frames occurs about 5-10% of the time.
 Gag includes the structural proteins matrix, capsid, and
nucleocapsid.
 Pol encodes the reverse transcriptase, endonuclease/integrase,
and the viral protease.
 Mutants that eliminated the -1 frameshift or made the Gag and
Pol ORFs in-frame both eliminated the production of infectious
virus.
 Thus, the ratio of Gag to Gag-Pol conferred by frameshifting is
critical for the viral life cycle.
While rare in cellular genes, -1 frameshifting
occurs in the E. coli DnaX gene
 The E. coli DnaX gene encodes two subunits of DNA
Polymerase III: the  subunit is the product of normal translation,
while the  subunit is derived by -1 frameshifting.
 Frameshifting occurs at the slippery sequence 5´-A AAA AAG3´ by simultaneous slippage of both the P and A site tRNAlys
species in the -1 direction.
 Frameshifting requires an SD-like element 10 nucleotides
upstream of the slippery sequence and a stem-loop structure 5
nucleotides downstream of the frameshift element.
 The extra distance to the SD element may enhance the
realignment (suggesting a pull-back mechanism).
-1 Frameshifting in the E. coli DnaX gene
DNA Pol III
10 nucleotides
Namy et al., Mol Cell 13: 157-1698 (2004)
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Ribosome Hopping
(aka Programmed Bypassing)
 Features of bacteriophage T4 gene 60
bypassing:
o matching GGA codons flanking an
optimally sized 50 nt coding gap
o a stop codon
o a stem loop structure
o a nascent peptide signal
 peptidyl-tRNA2Gly detaches from the
take-off site GGA, then pairs with the
landing-site GGA.
 Nearly all ribosomes initiate take off,
and ~50% resume translation in the
second ORF.
Herr et al., EMBO J 19: 2671-2680 (2000)
Features Important For Translational
Bypassing in Bacteriophage T4 Gene 60
 The nascent peptide signal is indicated by the yellow box.
 The matched take-off and landing codons, GGA, are shown in white letters in dark green
boxes.
 The UAG stop codon immediately 3' of the take-off site is in red letters next to the stop sign.
 Stop codons within the coding gap are overlined in red.
 Sequences that may be involved in base pairing in a potential extension of the stem-loop
are boxed in light green.
 A Shine–Dalgarno-like sequence is shown in the blue oval.
 The translational resume codon is indicated by the gray box.
Wills et al., EMBO J. 27: 2533-2544 (2008)
Current Model for T4 Gene 60 Programmed Bypassing
(A) The A-, P- and E-sites of the ribosome are filled with RNA
or shown by a dotted outline. The indirect influence of the
segment of the nascent peptide (yellow) on peptidyl-tRNA
anticodon: GGA 'take-off' codon (green flag) dissociation
is indicated by a dotted line.
The UAG (red flag) in the A-site causes a pause that
permits extra mRNA (dark blue) to start to enter the Asite, where it forms a structure diagrammed in (B). The
SD-like GAG sequence in the coding gap (dark blue
dashes in the mRNA) and the landing site codon, GGA
(white letters on green flag) are indicated.
(B) Intra-mRNA pairing drags mRNA initially from both the 5'
and 3' directions to allow formation of the 5' stem-loop.
Occupancy of the A-site by the mRNA structure precludes
entry by release factor 1 (pale green) and permits E-site
tRNA exit mediated by L9 (purple). Forward RNA
movement 'resolves' the structure in the A-site without
peptidyl-tRNA scanning.
(C) Return to linear mRNA and pairing of GAG (grey flag) 6 nt
5' of the end of the coding gap to the 3' end of 16S rRNA
(light blue) contributes to the initiation of peptidyl-tRNA
scanning and pairing to the landing site, GGA (green flag).
Standard decoding resumes at the adjacent 3' codon,
UUA (grey flag).
Wills et al., EMBO J. 27: 2533-2544 (2008)
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Incorporation of Selenocysteine, the 21st Amino
Acid, Occurs at In-Frame UGA Codons
 Whenever a stop codon enters the ribosomal A site, a competition
occurs between the class I release factor(s) and tRNA binding.
 For near- and non-cognate tRNAs, the release factor normally wins this
competition >99% of the time.
 Selenocysteine incorporation requires a dedicated tRNA that is cognate
for UGA codons
 Selenocysteine incorporation also requires a selenocysteine insertion
element (SECIS).
 In eubacteria, the specialized translation elongation factor SelB binds
both the SECIS just downstream of the SECIS and tRNAsec.
 In eukaryotes, the SECIS is located in the 3´-UTR of the mRNA.
Association of mSelB (also known as eEFsec) to the SECIS element
requires the adaptor protein SBP2.
Mechanism of selenocysteine incorporation
in prokaryotes and eukaryotes
 The translation elongation factor SelB (or mSelB) that delivers tRNAsecUCA to the A
site is functionally analogous eEF1A (but no known GTPase activity).
 One or two SECIS elements in the 3´-UTR of a eukaryotic mRNA can mediate
selenocysteine incorporation at many UGA codons in the mRNA.
 For example, expression of selenoprotein P in zebrafish requires the
reassignment of 17 UGA codons (!). This suggests that selenocysteine
incorporation can be very efficient.
Namy et al., Mol Cell 13: 157-1698 (2004)
Similar SECIS elements mediate selenocysteine incorporation
in prokaryotes and eukaryotes, but their location differ
Consensus
Hatfield & Gladyshev, Mol Cell Biol 22: 3565-3576 (2002)
Namy et al., Mol Cell 13: 157-1698 (2004)
The Sec tRNA Biosynthetic Pathway
in Archaea and Eukaryotes
Sec is synthesized on tRNAsec in three steps.
(1) The unacylated tRNAsec is charged by Ser tRNA synthetase with serine.
(2) The resulting Ser-tRNAsec is phosphorylated by phosphoseryl tRNA kinase (PSTK)
forming O-phosphoseryl-tRNAsec (Sep-tRNAsec).
(3) The phosphorylated intermediate is converted to the final product Sec-tRNAsec by SeptRNA:Sec tRNA synthase (SepSecS).
Yuan et al., FEBS Letters 584: 342-349 (2010)
Examples of selenocysteine-containing
proteins in animals
Many selenoproteins are found in animal cells. Consistent with their frequent occurrence,
selenoproteins are essential for mammalian development, since a tRNA(ser)sec knockout
mouse is embryonic lethal.
Hatfield & Gladyshev, Mol Cell Biol 22: 3565-3576 (2002)
Pyrrolysine, the 22 Amino Acid, is Encoded by
UAG Codons in Methanogenic Archaebacteria
 Pyrrolysine is an amide-linked 4substituted pyrroline-5-carboxylate
lysine derivative.
 It is found only in methanogenic
Archaebacteria. It occurs in proteins
that assist with the utilization of
methanogenic substrates like
trimethylamines.
 Each substrate requires activation by
a methyltransferase to generate
methane. All known methylamine
methyltransferase genes contain
pyrrolysine encoded at UAG codons.
Pyrrolysine, the 22 AA, is encoded by UAG
codons in methanogenic Archaebacteria
Little is currently known about the mechanism of pyrrolysine insertion at UAG codons, or
whether UAG codons can serve as stop codons in other genes. However, potential
pyrrolysine insertion (PYLIS) elements can be found 5-6 bases downstream of the sites of
insertion.
Namy et al., Mol Cell 13: 157-1698 (2004)
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Programmed Stop Codon Readthrough in Viral Genes
Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)
Programmed Stop Codon Readthrough in MuLV
Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)
Programmed Stop Codon Readthrough in MuLV
Requires a Downstream Pseudoknot
Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)
Pharmacological Suppression of Stop Codons
 Certain drugs can bind to the ribosome and reduce the ability
o Aminoglycosides
o PTC124
 May allow the treatment of a broad array of genetic diseases caused by
premature stop mutations
 What is the mechanism of aminoglycoside suppression?
Recall AA-tRNA proofreading during tRNA selection.
(Helix 34)
(Helix 18)
(Helix 44)
Ogle et al. (2002) Science 292:897-902.
(Helix 44)
Aminoglycosides Bind Helix 44 and Reduce
Ribosomal Fidelity During Translation
Aminoglycoside binding to Helix 44
leads to reduced elongation fidelity
(misreading) and less efficient
translation termination (readthrough).
Unbound
Paromomycin
bound
Yoshizawa et al, EMBO J. 17: 6437-6448 (1998); Ogle et al, Science 292:897-902 (2001)
Proximity of the Termination Complex to the Poly(A)
Tail is Important for Efficient Translation Termination
Normal Termination Codon (TC)
Premature Termination Codon (PTC)
PTC
eRF1
eRF3
TC
AUG
AUG
TC
eRF1
eRF3
eIF4E
eIF4G
PABPs
Normal Termination Codons:
Premature Termination Codons:
 The interaction between eRF3 and Poly(A)
Binding Protein (PABP) stimulates
termination.
 Interactions between eRF3 and PABP don’t
occur as efficiently.
 The eRF3/PABP interaction also promotes
ribosome recycling and stimulates
translational efficiency of the mRNA.
Adapted from Muhlemann, Biochem Soc Trans 36: 497-501 (2008)
 Stimulation of termination is also less
efficient, leading to a pause with the stop
codon in the ribosomal A site.
 Pausing is thought to make termination
complex more susceptible to readthrough of
the stop codon.
Clinical Applications of Stop Codon
Readthrough Therapies
 Small scale clinical trials for the pharmacological
suppression of premature stop mutations (stop
codon readthrough) have been carried out for
many diseases.
o
o
o
o
o
o
Cystic Fibrosis (Cl- transport disease)
Duchenne Muscular Dystrophy (dystrophin deficiency)
Factor VII deficiency (blood clotting disorder)
Hailey-Hailey disease (blistering skin disease)
Hemophilia A and B (blood clotting disorders)
McArdle Disease (muscle phosphorylase deficency)
What are Possible Complications Associated
With Stop Codon Readthrough Therapies?
 Readthrough of normal stop codons at the end of every gene.
 Nonsense-Mediated mRNA Decay (NMD) reduce mRNA
abundance, thus reducing the efficacy of readthrough
approaches.
 Ototoxicity and nephrotoxicity are associated with readthrough
agents like aminoglycosides
Other Recoding Therapies Considered
 Suppressor tRNAs to recode premature stop codons.
 Exon skipping induced by antisense oligonucleotides.
o Antisense oligonucleotides can interfere with exon recognition and
intron removal during pre-mRNA processing, and induce excision
of a targeted exon from the mature gene transcript.
o Targeted exon skipping of selected exons in the dystrophin gene
transcript can remove nonsense or frame-shifting mutations that
would otherwise have lead to Duchenne Muscular Dystrophy, the
most common childhood form of muscle wasting.
A
A
Bx
C
A
Bx
C
A
C
C
How Tolerant are Eukaryotic Cells to Recoding?
 Isolated frameshifting occurs in many contexts.
 Global readthrough induced by suppressor tRNAs or drugs (like
aminoglycosides or PTC124) are surprisingly well tolerated.
 Most amazing example of an organism surviving with global
recoding- many ciliated protozoa.
Phylogenetic Tree of eRF1 Molecules
and Associated Stop Codon Usage
Stop Codon Reassignment in Ciliates:
 UGA-only ciliates arose independently at
least 3 times: in Stylonichia+Oxytricha,
Loxodes, and Tetrahymena+Paramecium.
 UAA/UAG-specific ciliates arose at least
twice independently, in Euplotes and
Blepharisma.
Kim et al., Gene 346: 277 (2005)
Extremely High Rates of +1 Frameshifting
Occur in Euplotes Species
 Euplotes species use UAA and UAG as stop
codons, and have recoded UGA as a cysteine
codon.
 Most organisms have an extremely low incidence of
programmed translational frameshifting (e.g.,
frameshifting occurs in only 3 out of 6000 genes in
yeast, or 0.05%).
 8 out of 90 Euplotes genes sequenced to date
(~9%!) have in-frame +1 frameshift sites with similar
“shifty stop” slippery site (5´-AAA UAA A-3´). All but
one uses the UAA stop codon.
 Suggests high frameshifting is linked to the original
stop codon reassignment (when eRF1 lost UGA
recognition, UAA decoding also may have became
less efficient).
Klobutcher and Farabaugh, Cell 111:763-6 (2002)
Klobutcher, Euk. Cell 4: 2098-2105 (2005)
Euplotes octocarinatus:
UAA/UAG only
(UGA is a cys codon)
Recoding Mechanisms
 Ribosomal frameshifting
o +1 frameshifting
o -1 frameshifting
 Ribosome hopping
 Incorporation of unusual amino acids at stop codons
o Selenocysteine
o Pyrrolysine
 Stop codon readthrough
 Trans-translation
Rescue of a Prokaryotic Ribosome Stalled on a
truncated mRNA Molecule
 Transfer-messenger RNA (abbreviated
tmRNA) is a bacterial RNA molecule
with dual tRNA-like and mRNA-like
properties.
 The tmRNA forms a ribonucleoprotein
complex (tmRNP) together with SmpB
and EF-Tu.
 In trans-translation, tmRNA and its
associated proteins bind to bacterial
ribosomes which have stalled in the
middle of protein synthesis, (e.g. at the
end of an mRNA that has lost its stop
codon).
 The tmRNA adds a proteolysisinducing 11 AA tag on the unfinished
polypeptide, recycles the stalled
ribosome, and facilitates degradation
of the aberrant mRNA.
Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008)
Trans-Translation Removes All Components
of Stalled Translation Complexes
 tmRNA binds to SmpB and is aminoacylated by
alanyl-tRNA synthetase (AlaRS).
 EF-Tu in the GTP state binds to alanyl-tmRNA,
activating the complex for ribosome interaction
(box 1).
 The alanyl-tmRNA/SmpB/EF-Tu complex
recognizes ribosomes at the 3′end of an mRNA
and enters the A-site as though it were a tRNA.
 The nascent polypeptide is transferred to tmRNA,
and the tmRNA tag reading frame replaces the
mRNA in the decoding center. The mRNA is
rapidly degraded (box 2).
 Translation resumes, using tmRNA as a message,
resulting in addition of the tmRNA-encoded
peptide tag to the C terminus of the nascent
polypeptide. Translation terminates at a stop
codon in tmRNA, releasing the ribosomal subunits
and the tagged protein.
 Multiple proteases recognize the tmRNA tag
sequence and rapidly degrade the protein (box 3).
Nonstop and No-Go Translation
Complexes are Targeted For TransTranslation
 Errors or programmed events during
the normal process of protein
synthesis (box 1) produce a nonstop
translational complex when the
mRNA has no in-frame stop codon.
 Translation of the ribosome to the 3′
end of the mRNA generates a
substrate for trans-translation (box
2).
 Stalling during translation elongation
or termination results in a no-go
complex. The mRNA is cleaved in
the A-site or at the leading edge of
the ribosome, targeting the complex
for trans-translation.
Model For Trans-Translation
Regulation of the lacI mRNA
 Formation of the O1–LacI–O3
repression DNA loop causes
premature termination of LacI
transcription (resulting in mRNA
lacking a stop codon).
 Truncated lacI mRNAs are
recognized by the tmRNA system.
 Translation of defective LacI mRNA
is completed by trans-translation,
leading to destruction of both the
truncated mRNA and tagged LacI
protein.
Abo et al., EMBO J. 19: 3762-3769 (2000)