RECODING Myths in modern molecular

• The “universal ” is universal. • The genetic code is unambiguous. • are made only with 20 aa. • The “central dogma” describes the only flow of information. • Eukaryotic initiation only occurs in a cap- dependent manner

• Recoding mechanisms frequently provide exceptions RECODING MECHANISMS

• Ribosomal frameshifting . +1 frameshifting . -1 frameshifting • Ribosome hopping • readthrough • Incorporation of unusual amino acids at stop codons . . RECODING

am et al., Mol. , 2004 RIBOSOMAL FRAMESHIFTING

triplet code - three potential reading frames

• alternate mechanism of translation proteins encoded by two overlappingORFs • many retroviruses use framesifting for viral proteins • more common in prokarotes +1 Frameshifting

• EST3 encodes a subunit of telomerase with an internal programmed +1 frameshift site between ORF1 (93 aa) and ORF 2 (92aa) • The frameshift site has the slippery sequence 5´-CUU AGU U-3´. • AGU is encoded by a low abundance tRNA (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. • Other elements may be required.

yeast EST3 gene

Namy et al., Mol. Cell , 2004 +1 Frameshifting

• Extremely high rates of +1 frameshifting occur in Euplotes

• Euplotes use UAA and UAG as stop codons, and have recoded UGA as a cysteine codon

• translational frameshifting is rare ( e.g. 3 out of 6000 genes in yeast)

• in Euplotes 8 out of 90 have in-frame +1 frameshifts with “shifty stop” motif 5´-AAA UAA A-3´ (all but one uses the UAA stop codon).

• frameshifting probably linked to stop codon reassignment.

Euplotes: UAA/UAG only

Klobutcher and Farabaugh, Cell 111:763-6 (2002); Klobutcher, Euk Cell 4: 2098-2105 (2005) -1 frameshifting in retroviruses

(HIV)

Beet Western Yellow Virus (BWYV) -1 frameshift. Bases in red are conserved in luteoviruses. Frameshifting requires a 7 slippery site and a downstream pseudoknot.

Alam et al., Proc Natl Acad Sci USA 96: 14177-14179 (1999) -1 frameshifting in retroviruses

• Retroviral -1 frameshifting between the Gag and Pol ORFs occurs 5-10% of the time. • Ratio of Gag to Gag-Pol is critical for the viral life cycle. SELENOCYSTEINE the 21st aa

Incorporation of selenocysteine occurs at in-frame conserved UGA codons

• STOP UAG codon in the ribosomal A site • a competition between the class I release factor(s) (RFs) and near- cognate tRNAs (base pair at 2 of the 3 nts of the STOP codon). • RFs usually win 99.9% of the time • 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 , the SECIS is located in the 3´-UTR of the mRNA. Association of mSelB (eEFsec) to the SECIS element requires the adaptor SBP2. SELENOCYSTEINE INCORPORATION

(ser)sec • Translation elongation factor SelB (or mSelB) that delivers tRNA UCA to the A site is functionally analogous eEF1A (w/o 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. • Example: expression of P in zebrafish requires the reassignment of 17 UGA codons. Selenocysteine incorporation can be very efficient.

Namy et al., Mol Cell 13: 157-1698 (2004) SELENOCYSTEINE INCORPORATION

prokaryotic archaeal

eukaryotic

Berry, Nat Str Mol Biol, 2005 SECIS elements SELENOCYSTEINE INCORPORATION into a PROTEIN PYRROLYSINE the 22nd aa • encoded by UAG codons • found only in methanogenic Archaebacteria • pyrrolysine: amide-linked 4-substituted pyrroline-5-carboxylate lysine derivative. • occurs in proteins that assist with the utilization of methanogenic substrates like trimethylamines. • each substrate requires activation by a methyltransferase to generate methane • methylamine methyltransferase genes contain pyrrolysine encoded at UAG • insertion mechanism little known • potential pyrrolysine insertion PYLIS elements found 5-6 bases downstream of the sites of insertion.

Namy et al., Mol Cell 13: 157-1698 (2004) Reassignment of the Genetic Code: Genetic Code and its Variants

Blue letters: changes in mitochondrial lineages Bold letters: changes in nuclear lineages Blue boxes: codons that have changed only in mitochondria. Green boxes: codons that have changed both in mitochondrial and in nuclear lineages

Knight et al., Nature Rev Genetics 2: 49-58 (2001) PROTEIN DEGRADATION: UBIQUITINATION Ubiquitin

• biological regulation by proteolytic destruction of specific proteins • occurs in the cytoplasm and the nucleus PROTEIN DEGRADATION REGULATES: • Cell cycle • Differentiation & development • Extracellular effectors • Cell surface receptors & ion channels • DNA repair • Immune and inflammatory responses • Biogenesis of organelles

Proteins targeted by ubiquitin • cell cycle regulators • tumor suppressors and growth modulators • transcriptional activators and inhibitors • cell surface receptors • mutant and damaged proteins PROCESSES REGULATED BY UBIQUITINATION

Hochstrasser., Nature, 2011 UBIQUITIN • highly conserved 76 aa polypeptide (3 aa differences between yeast and human homologues) • C-Terminal Gly residue is activated via an ATP to form a thiol ester

1-MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF-45 Fission yeast 1-MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45 Green pea 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45 fruitfly 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76 46-AGKQLEDGRTLADYNIQKESTLHLVLRLRGG-76 human 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76 Ub chains

Ye and Rape., Nature Rev Cel Mol Biol, 2009 UBIQUITIN Genomic organization

Ubiquitin Ribosomal Ubiquitin Ubiquitin Ubiquitin protein

Transcription/Translation

Ub C-terminal hydrolysis

Ub Ub Ub Ub

Catalysis of isopeptide bond formation during ubiquitin chain synthesis. Conserved Asn in E2 interacts with the active-site Cys (with the donor ubiquitin) This stabilizes the oxyanion transition state of the nucleophilic attack by Lys of the acceptor ubiquitin

Ye and Rape., Nature Rev Cel Mol Biol, 2009 Ub linked to the ε -amino groups of Lys residues. Substrate amide linkage O Attachment of a single Ub C Ubiquitin NH is not a degradation signal NH Lys

Gly76

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

K48

K48-linked polyubiquitin chains act as a degradation signal Ub Chain assembly

Ub -C(O)OH

Ub -C(O)S- E1 Thioester linkage

Ub -C(O)S- E2

E3 Substrate Ub -C(O)NH Ub -C(O)NH Ub -C(O)-NH Amide bond linkage 3-step Ub conjugation Ub activating High energy thiol ester is formed enzyme E1 between C-terminal Gly of ubiqutin and a Cys in the E1 active site (ATP/AMP)

Ub conjugating Ub is transferred to a Cys of E2 forming enzymes E2 a new thiol ester

Ub ligase E3 Ub forms isopeptide bond between C- terminal Gly of Ub and ε-amino group of Lys on a target protein

Increasing level of regulatory specificity: E1: 1 E2: 10-12 (homologous family) E3: many and structurally unrelated

Classes of E3 ligases N-end Rule E3 • Specific E3 recognizes specific amino acids at the N-terminus of a protein • Following Met removal, N-terminal aa (Arg, Lys, His) are recognition signals RING finger class E3

HECT class E3

F class E3 TWO STEPS OF PROTEIN DEGRADATION via UBIQUITIN-PROTEASOME Covalent attachment of multiple ubiquitin (Ub) molecules to a substrate. Degradation of the tagged protein by the 26S proteasome (ubiquitin is recycled)

UBIQUITINATION- multiple Ubs in polyUb chains are linked via Lys48 of Ub Ye and Rape., Nature Rev Cel Mol Biol, 2009Biol, Mol Cel Rev Nature Rape., and Ye Ub chain synthesis

Ye and Rape., Nature Rev Cel Mol Biol, 2009 UBIQUITINATION

Brooks, WIREs RNA, 2010 PROTEIN DEGRADATION via UBIQUITINATION

ATP consuming process PROTEIN DEGRADATION via UBIQUITINATION 26S PROTEASOME

 degrades proteins in the cytosol, the nucleus, and the ER

 essential for the cell cycle (via the degradation of cyclins)

 essential for the immune response (via MHC-I peptides)

 a proven drug target (Velcade for multiple myeloma) Cellular targets

Cytoplasm

Lyzosome ER Lon/ PIM1

Nucleus

Degraded by other Degraded by the ubiquitin- proteases proteasome system. Role in the cell cycle

Nature Reviews in Cancer. Biol., vol 4, 349-360 (2004) Role in the immune system

Nature Reviews in Mol. Cell. Biol., vol 2, 179-188 (2001) 26S PROTEASOME Large- comparable to the ribosome Composition and subunit interaction studies by YTH and mass spec Structure by crystallography

proteasome

ribosomes

EM

Baumeister, W. (2005) Protein Science, 14 (1), 257-269 Proteasome: structure, activities 26S PROTEASOME 26S PROTEASOME

Bochtler et al., Annual Reviews of Biophysics and 1999 26S PROTEASOME

RNAse activity Brooks, WIREs RNA, 2010 26S PROTEASOME

Composed of 43 subunits with a molecular mass of about 2500 kD  Tunnel-like 20S catalytic core particle  Two 19S regulatory cap particles  Major substrates: polyubiquitinated proteins  Cleaves proteins in an ATP dependent manner

Fu et al, TiPlSci, 2010 PROTEIN DEGRADATION

Ub recycling by DUBs (deubiquitylating enzymes)

Protein degradation is regulated by protein degradation - degradation of Ub - degradation of E1-3 - degradation of proteasomal subunits

(e.g. during stress)

Wesissman et al, Nat Rev Mol Cell Biol, i, 2011 PROTEIN DEGRADATION inside the PROTEASOME

Hochstrasser., Nature, 2011 Nobel prize in chemistry, 2004

Aaron Ciechanover Avram Hershko Irwin Rose

"for the discovery of ubiquitin-mediated protein degradation" Co-translational protein and mRNA quality control

Lykke-Andersen and Bennett, JCB, 2014 Co-translational protein and mRNA quality control

NMD NGD

NSD RQC- ribosome QC complex Ltn1, Cdc48, Tae2, Rqc1 Ribosome release - Dom34/Hbs1/Rli1 Ubiquitin pathway components- Ltn1, Cdc48, Not4 Hel2, Asc1 (target nascent protein chain)

Lykke-Andersen and Bennett, JCB, 2014 PROTEIN DEGRADATION in the ER ERAD – ER ASSOCIATED DEGRADATION related to protein folding

Smith., Science, 2011 Venbar and Brodsky., Nat Rev Mol Cell Biol, 2008 UPR- UNFOLDED PROTEIN RESPONSE in ER STRESS Ire1- transmembrane serine-threonine kinase N-terminus in the ER lumen C-terminus in the cytosol.

• ufolded proteins accumulate in the ER • Ire1 oligomerizes • trans-autophosphorylates via the cytosolic kinase • activates the endonuclease in the tail domain • Ire1 endonuclease cleaves HAC1 mRNA removing the nonclassical (atypical splicing) • HAC1 exons ligated by tRNA ligase Rlg1 • induced HAC1 is translated • Hac1 - transcriptional activator • upregulates expression of UPR target genes (via binding to UPRE in promoters)

Venbar and Brodsky., Nat Rev Mol Cell Biol, 2008

UPR in yeast Three UPR pathways- three signal transcducer families

WALTER AND RON., Science 2011 SUMO Small Ubiquitin Related Modifier SUMO vs Ub

• SUMO does not have the Lys-48 found in Ub • SUMO does not make multi-chain forms • SUMO-1,2,3 are the mammalian forms • SUMO-1, 101 amino acids, C-terminal Gly, 18% identical to Ub SUMO Enzymes • SUMO-activating enzyme: heterodimer • SUMO-conjugating enzyme: Ubc9, an E2 enzyme (not Ub) • SUMO-ligase: E3 enzyme specific for sumoylation • E3-like proteins increase affinity between SUMO-Ubc9 and the target

SUMO Functions • Antagonistic role against ubiquitin: sumoylation often prevents ubiquitination and degradation (NF-κB pathway) • Protein translocation (Ran-GAP1/RanBp2) • Modulation of transcriptional activity (activates transcriptional activity of p53) • Subnuclear structure formation (protein targeting to nuclear bodies) Sumoylation and Desumoylation Cycle with E1-,E2-,E3-like Enzymes Proteins modified by SUMO

Protein Function RanGAP1 Nuclear transport PML Affected by translocations causing leukemias IB Inflammatory response p53 Tumor suppressor Mdm2 Regulates p53 Werner’s syndrome protein Mutations causing premature aging Septins Polarized cell growth and cytokinesis Proteins modified by SUMO SUMO vs Ub: Functions • Ub-proteasome pathway is essential for the degradation of damaged and regulatory proteins.

• Ub is activated in an ATP-consuming reaction as the E1-linked thioester.

• Ub is transferred in a transthiolation reaction to E2 proteins, which pass ubiquitin on to substrates with the help of E3 enzymes

• PolyUb (typically K48-linked) is a signal for degradation

• Degradation is carried out by the 26S proteasome (20S core)