Translational Recoding

1
Translational Recoding

• David Bedwell
• Post-Transcriptional Regulatory Mechanisms
  Advanced Course
• MIC759
• Oct 7, 2008

2
Myths in Modern Molecular Biology

• The universal genetic code is universal.
• The genetic code is unambiguous.
• All DNA encodes the information to make proteins
  with 20 amino acids.
• The central dogma of molecular biology
  describes the only flow of biological
  information.
• Eukaryotic translation initiation only occurs in
  a cap-dependent manner (a la Kozak).

3
Lecture Overview

• Background- what we need to know to understand
  many aspects of recoding
• Ribosomal frameshifting
• Ribosome hopping
• Incorporation of unusual amino acids at stop
  codons
• Stop codon readthrough
• Application of induced recoding to treat genetic
  diseases

4
Many Recoding Mechanisms Function During
Translation Elongation and Termination

• Frameshifting
• 1 frameshifting
• -1 frameshifting
• Ribosome Hopping
• Incorporation of unusual amino acids at in-frame
  stop codons
• Readthrough of premature stop codons
• Pharmacological
• Suppressor tRNAs

5
Translation Elongation
Translocation GTP hydrolysis
tRNA selection
Hybrid state
GTP hydrolysis proofreading
Peptide bond formation
EF1A EF-Tu EF1B EF-Ts EF2 EF-G
Merrick Nyborg, The Protein Synthesis
Elongation Cycle, In Translational Control of
Gene Expression (2000), CSHL Press, NY
6
tRNA Selection During Elongation
Ramakrishnan, Cell 108 557-572 (2002)
7
A Closer Look at the Fidelity of tRNA Selection
SSU Helix 44 is a Key Determinant of Fidelity
Helix 44 Decoding Site
Yusupov et al., Science 292 883-896 (2002)
Ramakrishnan, Cell 108 557-572 (2002)
8
Critical Nature of Residues A1492 and A1493 of
Helix 44 in Translational Fidelity
Ogle et al. (2002) Science 292897-902.
9
Aminoglycosides Bind Helix 44 and Reduce
Ribosomal Fidelity During Translation
Ogle et al, Science 292897-902 (2001)
10
Two Possible Outcomes When a Stop Codon Enters
the Ribosomal A Site
GTP
eRF1
eRF3
Termination (Normal Freq 99.9)
UAA
CAG
AUG
UAA
AAAAAAAAA
m7GpppG
A
P
Readthrough (Normal Freq 0.1)
Near-cognate AA-tRNA
11
Conditions that Shift the Balance Can Increase
the Frequency of Stop Codon Suppression
GTP
eRF1
eRF3
Termination (90-95)
UAA
CAG
AUG
UAA
AAAAAAAAA
m7GpppG
A
P
eEF1A
Readthrough (5-10)
Near-cognate AA-tRNA
GTP
12
Comparison of Eukaryotic and Prokaryotic
Termination Factors
13
Prokaryotic Translation termination
Zavialov et al., Cell 107 115-124 (2001)
14
Eukaryotic Translation Termination
Alkalaeva et al., Cell 125 1125-1136 (2006)
15
Recoding mechanisms include

• Ribosomal frameshifting
• 1 frameshifting
• -1 frameshifting
• Ribosome hopping
• Incorporation of unusual amino acids at stop
  codons
• selenocysteine
• Pyrrolysine
• Stop codon readthrough

16
RF2 expression in bacteria is subject to feedback
control by its own activity

• 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 good SD sequence 3 nucleotides upstream of
  the frameshift site (5-CUU UGA C-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
  SD-like element 3 nucleotides upstream (suggests
  a push-forward mechanism?).
• In this way, more RF-2 is made when there is not
  enough to rapidly terminate translation at the
  UGA stop codon.

17
1 Frameshifting Required for E. coli RF-2
Synthesis
18
Cellular Polyamine Levels Control Antizyme 1
Synthesis

• 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 ubiquitin-independent
  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.

19
1 Frameshifting in Antizyme Synthesis
Pseudoknot Structure
shifty stop slippery sequence (5-UCC UGA-3)
Namy et al., Mol Cell 13 157-1698 (2004)
20
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.

21
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)
22
-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 a 7
nucleotide slippery site and a downstream
pseudoknot.
Alam et al., Proc Natl Acad Sci USA 96
14177-14179 (1999)
23
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.

24
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 AAG-3 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).

25
-1 Frameshifting in the E. coli DnaX gene
DNA Pol III
10 nucleotides
Namy et al., Mol Cell 13 157-1698 (2004)
26
Recoding mechanisms include

• Ribosomal frameshifting
• 1 frameshifting
• -1 frameshifting
• Ribosome hopping
• Incorporation of unusual amino acids at stop
  codons
• selenocysteine
• Pyrrolysine
• Stop codon readthrough

27
Ribosome Hopping

• Bacteriophage T4 gene 60 bypassing requires
• - matching GGA codons flanking an optimally sized
  50 nt coding gap
• - a stop codon
• - a stem loop structure
• a nascent peptide signal.
• In the current model, peptidyl-tRNA2Gly detaches
  from the take-off site GGA, scans the mRNA as it
  slides through the P-site, then pairs with the
  landing-site GGA. Nearly all ribosomes initiate
  scanning, and 50 resume translation in the
  second ORF.

Herr et al., EMBO J 19 2671-2680 (2000)
28
Recoding mechanisms include

• Ribosomal frameshifting
• 1 frameshifting
• -1 frameshifting
• Ribosome hopping
• Incorporation of unusual amino acids at stop
  codons
• selenocysteine
• Pyrrolysine
• Stop codon readthrough

29
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 near-cognate tRNAs (that
  can base pair at 2 of the 3 nucleotides of the
  stop codon).
• The release factor normally wins this competition
  gt99 of the time, but this efficiency can be
  reduced by the sequence context around the stop
  codon, the relative level of the release factor,
  and the presence of downstream elements that can
  stimulate suppression.
• Selenocysteine incorporation requires a
  selenocysteine insertion element (SECIS).
• In eubacteria, the specialized translation
  elongation factor SelB binds both the SECIS just
  downstream of the SECIS and tRNA(ser)sec.
• 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.

30
Mechanism of selenocysteine incorporation in
prokaryotes and eukaryotes

• The translation elongation factor SelB (or mSelB)
  that delivers tRNA(ser)secUCA 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)
31
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)
32
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)
33
Pyrrolysine, the 22 AA, is encoded by UAG codons
in methanogenic Archaebacteria
Pyrrolysine is an amide-linked 4-substituted
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.
34
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. 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)
35
Recoding mechanisms include

• Ribosomal frameshifting
• 1 frameshifting
• -1 frameshifting
• Ribosome hopping
• Incorporation of unusual amino acids at stop
  codons
• selenocysteine
• Pyrrolysine
• Stop codon readthrough

36
Programmed stop codon readthrough in viral genes
Beier Grimm, Nucl. Acids Res 29 4767-4782
(2001)
37
Programmed stop codon readthrough in MuLV
Beier Grimm, Nucl. Acids Res 29 4767-4782
(2001)
38
Programmed stop codon readthrough in MuLV
requires a downstream pseudoknot
Beier Grimm, Nucl. Acids Res 29 4767-4782
(2001)
39
Pharmacological suppression of stop codons

• Certain drugs can bind to the ribosome and reduce
  the ability
• Aminoglycosides
• PTC124
• May allow the treatment of a broad array of
  genetic diseases caused by premature stop
  mutations
• What is the mechanism of aminoglycoside
  suppression?

40
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).
Yoshizawa et al, EMBO J. 17 6437-6448 (1998)
Ogle et al, Science 292897-902 (2001)
41
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.
• 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)

42
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)
• Other toxicities associated with readthrough
  agents like aminoglycosides

43
Other Recoding Therapies Considered

• Suppressor tRNAs to recode premature stop codons.
• Exon skipping induced by antisense
  oligonucleotides.
• 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.
• 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.

44
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.

45
Phylogenetic Tree of eRF1 Molecules and
Associated Stop Codon Usage
Stop codon reassignment in cilates UGA-only
ciliates arose independently at least 3 times in
(StylonichiaOxytricha), in Loxodes, and in
(Tetrahymena Paramecium). UAA/UAG-specific
ciliates arose at least twice independently, in
Euplotes and in Blepharisma.
Kim et al., Gene 346 277 (2005)
46
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 111763-6 (2002)
Klobutcher, Euk Cell 4 2098-2105 (2005)