•  The “universal genetic code” is universal.

RECODING

Myths in modern molecular biology

•  The “universal genetic code” is universal.

•  The genetic code is unambiguous.

•  Proteins are made with 20 aa.

•  The “central dogma” describes the only flow of information.

•  Eukaryotic translation initiation only occurs in a cap- dependent manner

•  Recoding mechanisms frequently provide exceptions

RECODING MECHANISMS

•  Ribosomal frameshifting

§  +1 frameshifting

§  -1 frameshifting

•  Ribosome hopping

•  Stop codon readthrough

•  Incorporation of unusual amino acids at stop codons

§  selenocysteine

§  pyrrolysine

•  EST3 encodes a subunit of telomerase

•  Synthesis of the full-length protein requires an internal programmed +1 frameshift 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 tRNA^leu in the P site can undergo +1 slippage to the overlapping UUA codon.

+1 Frameshifting

Namy et al., Mol. Cell , 2004

yeast EST3 gene

SELENOCYSTEINE the 21st aa

Incorporation of selenocysteine occurs at in-frame conserved UGA codons

•  STOP UGA 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 eukaryotes, the SECIS is located in the 3´-UTR of the mRNA.

Association of mSelB (eEFsec) to the SECIS element requires the adaptor protein SBP2.

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

•  Translation elongation factor SelB (or mSelB) that delivers tRNA^(ser)sec_UCAto 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.

•  In eukaryotes association of SelB to the SECIS element requires the adaptor protein SBP2

•  Example: expression of selenoprotein P in zebrafish requires the reassignment of 17 UGA codons.

Selenocysteine incorporation can be very efficient.

SELENOCYSTEINE INCORPORATION

prokaryotic archaeal

eukaryotic

Berry, Nat Str Mol Biol, 2005

SELENOCYSTEINE INCORPORATION

SECIS elements

SELENOCYSTEINE INCORPORATION

PYRROLYSINE the 22nd aa

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

•  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.

PROTEIN DEGRADATION:

UBIQUITINATION

PROTEASOME

Regulation by proteolytic destruction of specific proteins

Occurs in the cytoplasm and the nucleus

"for the discovery of ubiquitin-mediated protein degradation"

Nobel prize in chemistry, 2004

Aaron Ciechanover Avram Hershko Irwin Rose

REGULATES:

•  Cell cycle

•  Differentiation & development

•  Extracellular effectors

•  Cell surface receptors & ion channels

•  DNA repair

•  Immune and inflammatory responses

•  Biogenesis of organelles

PROTEIN DEGRADATION

Proteins targeted by ubiquitin

•  cell cycle regulators

•  tumor suppressors and growth modulators

•  transcriptional activators and inhibitors

•  cell surface receptors

•  mutant and damaged proteins

Hochstrasser., Nature, 2011

PROCESSES REGULATED BY

UBIQUITINATION

UBIQUITIN

•  highly conserved 76 aa polypeptide

•  C-Terminal Gly residue is activated via an ATP to form a thiol ester

1-MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76

46-AGKQLEDGRTLADYNIQKESTLHLVLRLRGG-76 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76 46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76

Fission yeast

human Green pea fruitfly

Ubiquitin Ubiquitin Ubiquitin Ubiquitin Ribosomal protein

Transcription/Translation

Ub C-terminal hydrolysis

Ub Ub Ub Ub

Genomic organization

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

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

UBIQUITIN

Ub chains

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

Function of Ub chains

Rape., Nat Rev Cel Mol Biol, 2017; Buetow and Huang, Nat Rev Cel Mol Biol, 2016

Function of Ub chains

Kwon and Ciechanover., TiBS, 2017

C

Lys

O

NH NH

Gly76 Ubiquitin

Substrate

amide linkage Ub is linked via isopeptide bond between COO

of its Gly76 to the ε-NH

groups of Lys residues.

Ub can be also linked to the N-terminal Met or other

residues: Cys, Thr or Ser.

Attachment of a single Ub is not a degradation signal

K48-linked polyubiquitin chains act as a degradation signal

UBIQUITINATION

MQI FVKTL TG KTI TL EVEPS DTI ENV KAKI QDKEGI PPDQ QRL I FAGKQL EDGRTL SDYN I QKESTL HL V L RL RGG

K48

-C(O)OH Ub

-C(O)S- E1 Ub

-C(O)S-

Ub E2

E3

-C(O)-NH Ub

-C(O)NH Ub

-C(O)NH Ub

Substrate

Thioester linkage

Amide bond linkage

Ub Chain assembly

3-step Ub conjugation

Ub activating enzyme E

High energy thiol ester is formed

between C-terminal Gly of ubiqutin and a Cys in the E

active site (ATP/AMP)

Ub conjugating enzymes E

Ub is transferred to a Cys of E

forming a new thiol ester

Ub ligase E

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

RING finger class 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

N-end Rule E3 Classes of E3 ligases

HECT class E3

F class E3

UBIQUITINATION

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

Covalent attachment of multiple ubiquitins (Ub) to a substrate via Lys48 in Ub

Elongation Initiation

Ub activating enzyme

Ub conjugating enzyme

Ub ligase

Brooks, WIREs RNA, 2010

UBIQUITINATION

PROTEIN DEGRADATION via UBIQUITINATION

Tagged proteins are degraded by the 26S proteasome Ubiquitin is recycled

ATP consuming process

u 

degrades proteins in the cytosol, the nucleus and the ER

u 

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

u 

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

u 

a proven drug target (Velcade for multiple myeloma)‏

26S PROTEASOME

Cellular targets

Lyzosome

Nucleus ER Cytoplasm

Lon/

PIM1

Degraded by the ubiquitin- proteasome system.

Degraded by other

proteases

Fu et al, TiPlSci, 2010

²  Composed of 43 subunits with a molecular mass of about 2500 kD

²  Tunnel-like 20S catalytic core particle

²  Two 19S regulatory cap particles

²  Major substrates: polyubiquitinated proteins

²  Cleaves proteins in an ATP dependent manner

26S PROTEASOME

26S PROTEASOME PTM

PTM- Post Translational Modifications

Livneh et al, Cell Res , 2016

Baumeister, W. (2005) Protein Science, 14 (1), 257-269

26S PROTEASOME

ribosomes

proteasome

EM

Large- comparable to the ribosome

Composition and subunit interaction studies by YTH and mass spec

Structure by crystallography and CryoEM

Proteasome: structure, activities

Bochtler et al., Annual Reviews of Biophysics and Biomolecular Structure 1999

26S PROTEASOME

26S PROTEASOME

Brooks, WIREs RNA, 2010 RNAse activity

26S PROTEASOME

Hochstrasser., Nature, 2011

PROTEIN DEGRADATION inside the

PROTEASOME

Greene et al., Curr Op Struct Biol , 2020

PROTEIN DEGRADATION inside the PROTEASOME

CryoEM

Wesissman et al, Nat Rev Mol Cell Biol, i, 2011

PROTEIN

DEGRADATION

Protein degradation is regulated by protein degradation

- degradation of Ub - degradation of E1-3 - degradation of

proteasomal subunits (e.g. during stress)

Ub recycling by DUBs (deubiquitylating enzymes)

Co-translational protein and mRNA QC

Lykke-Andersen and Bennett, JCB, 2014

Lykke-Andersen and Bennett, JCB, 2014

NMD

NSD

NGD

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)

Co-translational protein and mRNA QC

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

•  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)

SUMO Functions

Sumoylation and Desumoylation Cycle

with E1-,E2-,E3-like Enzymes

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)

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