W10/11: Translation

elongation speed: 14 1/s

error frequency: 10^(-3) - 10^(-8)

• RNA molecule with defined secundary structure (cloverleaf)

• 70-80 bp long

• 5’ phosphorylated

• 3’ amino acid binding site (ACC)

• anticodon on “middle” loop

Isoacceptors: tRNAs that read different codons but carry the same amino acid (read synonymous codons)

• different codons coding for the same amino acid can have different abundances; use of codon can influence translational flow and therefore protein folding (—> it is relevant which codon is used, not just which amino acid results from it)

• codon usage can be different in different organisms and might be important to consider when expressing recombinant proteins in a different organism (harmonization)

Isodecoders (eukaryotes): tRNAs that read the same codons and carry the same amino acid, but have sequence deviations other than in the anticodon

• conserved in most organisms, but can differ (e.g., mitochondria)

Aminoacyl tRNA Synthetases

1. Activation of the amino acid (ATP) by binding AMP.

2. tRNA binds, AMP is exchanged with tRNA

Specificity of ARSs

• tRNA recognition:

  • large interaction site between ARS and tRNA (anti-codon and other specific sites)

    • high fidelity

• amino acid recognition

  • specific binding pocket

  • kinetic discrimination (e.g., in cases such as Ile/Val)

    1. pre-transfer: amino acids can be hydrolyzed by the synthetase before transfer to the tRNA; this is faster for false amino acids

    2. post-transfer: amino acid can be cleaved from the tRNA in the editing site; this is faster for false amino acids

Large subunit (50S)

• 2 RNAs

• 34 proteins

Small subunit (30S)

• 1 RNA

• 21 proteins

General:

• proteins are rather located around the surface, RNAs at the subunit interface

• L12 stalk(50S)

  • ribosomal protein, 4-8 copies

  • “fishing rod” functionality - recruits IFs and EF, interacts with GTPases

• SRL region (near L12, 50S)

  • activation of GTPases after recruitment

Multiple ribosomes translating the same mRNA at the same time

• minimal spacing between ribosomes: 30nt

• average spacing between ribosomes: 80nt

Polysomes attenuate effects of secondary structures of mRNAs.

Polysomes have a potential effect on protein folding and polymerization.

Initiation

• IF1, IF2, IF3

Elongation

• EF-Tu

• EF-G

• EF-P

• SelB

Termination

• RF1, RF2, RF3

Ribosome recycling

• RRF, EF-G, IF3

Differences in eukaryotic translation:

• more complex, more factors involved

• no co-transcriptional translation possible (nucleus)

• no Shine-Dalgarno sequence

  • recognition at 5’-cap

• 3’-poly-A tail and 5’-cap

• mRNA is circularized

• mRNA is monocistronic

• …

Elongation is comparable in eukaryotes and prokaryotes.

• joining of 50S subunit leads to GTP hydrolysis

Control elements

• interaction between Shine-Dalgarno sequence (mRNA) and anti-Shine-Dalgarno sequence (ribosome)

• length of spacer (region between SD sequence and start codon)

• start codon recognition

• secondary structures at the ribosomal binding site

• AU-rich region (3’-UTR) in mRNA interacts with ribosome

1. aa-tRNA selection (decoding)

• EF-Tu, GTP and aa-tRNA form complex

  • bound by L12-stalk

• recognition of tRNA, binding to ribosome (A-site)

  • GTP hydrolysis (GTPase domain comes close to the GTPase-activating center of the ribosome)

• EF-Tu-GDP dissociates and is recycled

1. Peptide bond formation

• peptide chain from tRNA in P-site is transferred to aa bound to the tRNA in P-site

• catalized by rRNA

  
  

1. Translocation

• EF-G + GTP complex binds to the ribosome

• GTP hydrolysis -> translocation

• EF-G + GDP dissociate and are recycled

Fidelity paradox: The energy difference between correct and false tRNA-binding is only 10-150 fold —> not enough to discriminate

Therefore: Kinetic discrimination

• general principle: reaction is faster for correct pairing

  • Induced Fit!

• happens 2 times during decoding:

  • initial selection: reaction before GTP hydrolysis (irreversible)

  • proofreading: aa-tRNA release is faster for false aa than aa-tRNA accomodation

(nicht lernen, vielleicht Begriffe)

“The ribosome provides a network of interactions that position the reactants and the transition state intermediate, shields them from bulk water and provides favorable electrostatic environment.”

EF-P is necessary for peptide bond formation with prolines (stiffer than other aa, reacts very slowly otherwise).

PRE complex: ribosome after peptide transfer, still in “classical” conformation

PRE* complex: subunits are rotated against each other, tRNAs are in “hybrid” state

POST complex: ribosome after translocation, again in “classical” conformation, no rotation

EF-G promotes switch from PRE* to POST by GTP hydrolysis (loaded spring mechanism).

• Translation is accelerated by EF-G but does take place without it, too.

• EF-G is a molecular motor

  • “promoting unidirectional motion powered by external energy input”

• EF-G prevents reverse movement (doorstop-like)

• EF-G stabilizes codon-anticodon interaction (reading frame maintenance)

1. Peptide bond formation (elongation)

2. Peptidyl-tRNA hydrolysis (termination)

• recognition of stop-codons by RF1 or RF2

• GTPase RF3 promotes release of RF1/2 (?)

Factors influencing translational flow

• mRNA properties (secondary structures, rare codons, certain sequences)

• tRNA availability and properties

• polysome formation

• occurrence of Pro (slow peptide-bond formation) or poly-Lys

• interactions between Arg/Lys with peptide exit tunnel

Slower translation can have effects on protein folding.

• Peptide exit channel of the ribosome

  • folding due to shape of and interactions with the tunnel

  • outcome depends on translation speed (how much time the protein spends inside the exit channel)

• trigger factors

  • chaperone-assisted folding outside the ribosome

Other proteins:

• peptide deformylase (fMet cleavage)

• signal recognition particle (protein targeting)

• metionine aminopeptidase (maturation)

• in addition to Watson-Crick base pairing between codon and anticodon, so-called wobble base pairing is possible as well

  • example: UUU and UUC code for Phe (both are translated with the same tRNA)

  • especially the last (5’) base of the anticodon is prone to wobble base pairing (less spacially confined)

    
    

GTPases - Overview

General roles of GTPases:

• fidelity (kinetic discrimination): IF2, EF-Tu

• enhancement of translocation rate: EF-G

ATP

• important for aa binding to tRNA (ARSs)

Differences prokaryotes/eukaryotes:

• pro: 70S = 50S + 30S; eu: 80S = 60S + 40S

• pro: coupling of translation and transcription

• fundamentally similar, differences in the details (mainly initiation)

Mitochondrial ribosomes:

• smaller than bacterial ribosomes

• smaller rRNA content

• peptide bond formation (rRNA as catalyst)

• peptide release from tRNA (ribosome and release factors are involved)

Mostly yes, but there are exceptions

• e.g. mitochondria

  • slightly different codon usage, e.g. stop-codons

Redefinition is a type of recoding.

Recoding

• Redefinition: certain codons translate to other aa than usual (e.g. selenocysteine or pyrollysine); StopGo-translation

• Frameshifting: the reading frame is shifted +1/-1

  • occurs at “slippery sides” (multiple Lys after each other) followed by secondary elements in the mRNA

  • does not happen in 100% of cases

  • important for viruses (confined genome size), e.g. HIV and coronavirus

• Bypassing: hairpin-structure in mRNA, ribosome stops on Lys-codon, moves along the mRNA up to 60 nucleotides, lands on Lys.

Why is recoding used?

• larger number of proteins from the same genome (important for viruses)

• expression regulation (?)

• different codon usage in original organism and host

  • result: false aa sequence

• different frequency of codons

  • result: different translation speed, may lead to misfolding of proteins

• lack of chaperones in host

  • result: protein might not fold properly on its own

Eukaryotes:

• much more factors contributing to initiation

• formation of pre-initiation complex first (eIFs and mRNA), then binding of the pre-initiation complex and tRNA+factors to the small subunit