2 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.
3 Lecture OverviewBackground: examples of recoding that will allow us to understand more translation.Ribosomal frameshiftingRibosome hoppingIncorporation of unusual amino acids at stop codonsStop codon readthroughPharmacological suppressionSuppressor tRNAsSwitching mRNA templates during translationClinical implications:Induced recoding may help treat many genetic diseasesPrevention of recoding may help treat some infectious diseases.
4 Comparison of Eukaryotic and Prokaryotic Elongation Factors ProkaryotesEF-Tu (GTPase)EF-Ts (GEF)EF-G (GTPase)EukaryoteseEF1A (GTPase)eEF1B (GEF)eEF2 (GTPase)
5 Translation Elongation EF2:GDPEF2:GTPEF1A:GDPEF1BGDPEF1A:EF1BGTPEF1A:GTPAA-tRNAAA-tRNA:EF1A:GTPa)b)c)d)e)Translocation & GTP hydrolysistRNA selectionHybrid stateGTP hydrolysis & proofreadingPeptide bond formationMerrick & Nyborg, The Protein Synthesis Elongation Cycle, In Translational Control of Gene Expression (2000), CSHL Press, NY
6 tRNA Selection During Elongation Recoding can occur hereRamakrishnan, Cell 108: (2002)
8 Prokaryotic Translation Termination Zavialov et al., Cell 107: (2001)
9 Eukaryotic Translation Termination Alkalaeva et al., Cell 125: (2006)
10 A Closer Look at the Fidelity of tRNA Selection: SSU Helix 44 is a Key Determinant of Fidelity “Decoding Site”Ramakrishnan, Cell 108: (2002)Yusupov et al., Science 292: (2002)
11 Critical Nature of A1492 and A1493 in Helix 44 of 16S rRNA in Translational Fidelity Ogle et al. (2002) Science 292:
12 Aminoglycosides Bind Helix 44 and Reduce Ribosomal Fidelity During Translation UnboundParomomycin bound- tRNA+ tRNAOgle et al, Science 292: (2001)
13 A Competition Occurs When Any Codon Enters the Ribosomal A Site Example: Stop Codon Recognition)GTPeRF1eRF3Termination(Normal Freq ~99.9%)m7GpppGAUGCAGUAAUAAAAAAAAAAAPAeEF1AGTPCompetitions:Stop codon vs. near-cognate codonsSense codon vs. near-cognate codonsReadthrough(Normal Freq ~0.1%)Near-cognate AA-tRNA
14 Various Conditions can Shift This Balance to Increase the Frequency of Mis-Incorporation GTPeRF1eRF3Termination(90-95%)m7GpppGAUGCAGUAAUAAAAAAAAAAAPAA key recoding Inducer is pausing caused by:Hungry codonsmRNA structuresRecoding enhanced by:Slippery Sites in mRNADedicated factorseEF1AReadthrough(5-10%)Near-cognate AA-tRNAGTP
15 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
16 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
17 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.
18 +1 Frameshifting Required for E. coli RF-2 Synthesis **Namy et al., Mol Cell 13: (2004)***E. coliS30RRLDonley & Tate, Proc Biol Sci 244: (1991)
19 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 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.
20 +1 Frameshifting in Antizyme Synthesis PseudoknotStructurepoorly defined 5´ stimulatory sequence“shifty stop”slippery sequence(5´-UCC UGA-3´)Namy et al., Mol Cell 13: (2004)
21 +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.
22 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: (2004)
23 -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 sitedownstream pseudoknotAlam et al., Proc Natl Acad Sci USA 96: (1999)
24 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.
25 While rare in cellular genes, -1 frameshifting occurs in the E While rare in cellular genes, -1 frameshifting occurs in the E. coli DnaX geneThe 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).
26 -1 Frameshifting in the E. coli DnaX gene DNA Pol III10 nucleotidesNamy et al., Mol Cell 13: (2004)
27 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
28 (aka Programmed Bypassing) Ribosome Hopping(aka Programmed Bypassing)Features of bacteriophage T4 gene 60 bypassing:matching GGA codons flanking an optimally sized 50 nt coding gapa stop codona stem loop structurea nascent peptide signalpeptidyl-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: (2000)
29 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: (2008)
30 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 A- site, 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: (2008)
31 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
32 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 codonsSelenocysteine 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.
33 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: (2004)
34 Similar SECIS elements mediate selenocysteine incorporation in prokaryotes and eukaryotes, but their location differConsensusHatfield & Gladyshev, Mol Cell Biol 22: (2002)Namy et al., Mol Cell 13: (2004)
35 The Sec tRNA Biosynthetic Pathway in Archaea and Eukaryotes Sec is synthesized on tRNAsec in three steps.The unacylated tRNAsec is charged by Ser tRNA synthetase with serine.The resulting Ser-tRNAsec is phosphorylated by phosphoseryl tRNA kinase (PSTK) forming O-phosphoseryl-tRNAsec (Sep-tRNAsec).The phosphorylated intermediate is converted to the final product Sec-tRNAsec by Sep- tRNA:Sec tRNA synthase (SepSecS).Yuan et al., FEBS Letters 584: (2010)
36 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: (2002)
37 Pyrrolysine, the 22 Amino Acid, 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.
38 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: (2004)
39 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
40 Programmed Stop Codon Readthrough in Viral Genes Beier & Grimm, Nucl. Acids Res 29: (2001)
41 Programmed Stop Codon Readthrough in MuLV Beier & Grimm, Nucl. Acids Res 29: (2001)
42 Programmed Stop Codon Readthrough in MuLV Requires a Downstream Pseudoknot Beier & Grimm, Nucl. Acids Res 29: (2001)
43 Pharmacological Suppression of Stop Codons Certain drugs can bind to the ribosome and reduce the abilityAminoglycosidesPTC124May allow the treatment of a broad array of genetic diseases caused by premature stop mutationsWhat is the mechanism of aminoglycoside suppression?Ogle et al. (2002) Science 292:(Helix 44)(Helix 18)(Helix 34)Recall AA-tRNA proofreading during tRNA selection.
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).UnboundParomomycin boundYoshizawa et al, EMBO J. 17: (1998); Ogle et al, Science 292: (2001)
45 Proximity of the Termination Complex to the Poly(A) Tail is Important for Efficient Translation TerminationNormal Termination Codon (TC)eRF1eRF3eIF4EeIF4GPABPsAUGTCPremature Termination Codon (PTC)eRF1eRF3AUGTCPTCNormal Termination Codons:The interaction between eRF3 and Poly(A) Binding Protein (PABP) stimulates termination.The eRF3/PABP interaction also promotes ribosome recycling and stimulates translational efficiency of the mRNA.Premature Termination Codons:Interactions between eRF3 and PABP don’t occur as efficiently.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.Adapted from Muhlemann, Biochem Soc Trans 36: (2008)
46 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)
47 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
48 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.ABCxAC
49 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.
50 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)
51 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).Euplotes octocarinatus:UAA/UAG only(UGA is a cys codon)Klobutcher and Farabaugh, Cell 111:763-6 (2002)Klobutcher, Euk. Cell 4: (2005)
52 Recoding Mechanisms Ribosomal frameshifting Ribosome hopping Incorporation of unusual amino acids at stop codonsSelenocysteinePyrrolysineStop codon readthroughTrans-translation
54 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).
55 Nonstop and No-Go Translation Complexes are Targeted For Trans-Translation 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.
56 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: (2000)
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