Protein Synthesis: Translation

• Chapter 18 discusses Protein Synthesis

Translation Initiation in Bacteria

• Translation in bacteria begins at ribosome binding sites (RBS).
• Prokaryotic mRNAs generally are polycistronic (containing 2+ ORFs).
• Ribosomes assemble directly at the RBS.
• The Shine-Dalgarno sequence is the same as the RBS.
• Each ORF on bacterial polycistronic mRNAs may have its own RBS.
• The stop codon of one ORF can overlap with the start codon of the next, which is known as translational coupling.
• In translational coupling, the ribosome terminates polypeptide synthesis at the stop codon, then backs up one nucleotide and starts another round of elongation.

Translation Initiation in Eukaryotes

• Ribosomes begin assembly at the 5' CAP and scan downstream for a Kozak consensus sequence.
• Eukaryotic mRNAs are typically monocistronic (containing one ORF).
• Ribosome small subunit first associates at the 5' CAP.
• The Kozak Sequence is GCCRCCAUGG for vertebrates.

Ribosome Structure

• Ribosomes are composed of large and small subunits.
• Ribosomes consist of ~60% RNA and ~40% protein.
• Eukaryotic ribosomes are 80S, made of 40S and 60S subunits.
• Prokaryotic ribosomes are 70S, made of 30S and 50S subunits.
• Units of analytical centrifugation are called Svedbergs (S).
• Larger molecules sediment faster and have higher S values.
• Polysomes are multiple ribosomes attached to a single mRNA.
• One ribosome covers ~80 nucleotides of mRNA.
• A 1000 nucleotide ORF (~36kD protein) could have ~12 ribosomes, given that an average amino acid is ≈110 Da.

tRNA Binding Sites

• Ribosomes have three tRNA binding sites: E (exit) site, P (peptidyl-tRNA) site, and A (aminoacyl-tRNA) site.
• The large ribosomal subunit (SU) contains a peptidyl transferase center.
• The small SU contains binding sites for mRNA and tRNAs which is known as the decoding center.
• Ribosomes have a protein exit tunnel wide enough for an alpha helix.
• Further protein folding has to wait until translation is finished.

Translation Cycle

• Translation involves cycles of initiation, elongation, and termination.
• Activation of amino acids happens when tRNA is aminoacylated.
• In initiation, mRNA and aminoacylated tRNA bind to the small ribosomal subunit, after which the large subunit also binds.
• In elongation, successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until the ribosome reaches a stop codon.
• Termination happens when a stop codon is encountered, causing translation to stop.
• The mRNA and protein dissociate, and the ribosomal subunits are recycled.
• Protein folding is the last step.

Prokaryotic Initiation

• The 30S subunit binds IF-1 and IF-3, then binds the mRNA.
• IF3 blocks the large subunit and prevents ribosome assembly.
• IF1 blocks tRNA from A site during initiation phase.
• The Shine-Dalgarno sequence binds near 3' end of 16S mRNA to properly align the P site with start codon.
• The first tRNA is always Met-tRNAfmet, N-formyl methionine after this special Met-tRNA is charged.
• Met-tRNAfmet is escorted to the P-site by IF-2-GTP.
• The fMet-tRNA fMet, accompanied by IF-2, base-pairs with the start codon.
• When everything is properly in place, GTP on IF2 is hydrolyzed and IF factors are released.

Eukaryotic Initiation

• Multiple elFs on the 40S ribosome subunit block ribosome assembly or serve as scaffolding for recruitment of other factors.
• elF1A blocks tRNA from A site during initiation phase.
• elF2-GTP escorts Met-tRNAiMet (initiator tRNA) to the P site and elF5B-GTP helps it associate stably.
• The 43S preinitiation complex assembles before association with mRNA.
• elF4F is a "cap binding protein" that binds the 5' CAP of mRNA.
• elF4F recruits the 43S complex to the 5' end of mRNA.
• elF4F also acts as an RNA helicase, using ATP hydrolysis to thread mRNA through 43S until the anticodon of P-site Met-tRNAiMet pairs with the start codon.
• When the start codon is reached, GTP is hydrolyzed, and elF factors are released.
• A 60S ribosomal subunit can then associate.
• Assembly of the 80S complex completes the initiation phase.
• elF4F associates with the 5'CAP AND the poly-A tail, resulting in mRNA circularization, more efficient translation, and greater resistance to degradation.
• Some viruses and a few cellular mRNAs involved in viral defense and stress responses can translate mRNAs that lack a 5' cap, this is made possible by internal ribosome entry sites (IRES).

Elongation (Bacteria)

• EF-Tu-GTP (tRNA chaperone) escorts a charged tRNA to the A-site
• Correct tRNA base-pairing happens when the anticodon pairs with the codon.
• GTP hydrolysis results in EF-Tu release.
• EF-Tu-GDP release allows tRNA in A site to rotate and position the amino acid of the P-site in close proximity to the amino acid in the A site.
• EF-Tu-GDP gets recycled by GTP exchange factor (EF-Ts).
• The ribosome is a ribozyme since the 50S subunit alone can catalyze the peptidyl transferase reaction and only RNA components are essential for catalytic activity.
• EF-G (translocase) helps the ribosome move to the next codon after peptide bond formation, and It does so by mimicking an aminoacyl-tRNA bound to EF-Tu.
• EF-G-GTP(tRNA mimic) binds to the A-site and GTP hydrolysis triggers the translocation of tRNAs.
  • The A-site tRNA plus the amino acid chain translocates to the P site.
  • The P-site tRNA moves to the E site.
• Base-pairing with mRNA is maintained, so ribosome translocates too
• EF-G-GDP dissociates from the A-site
• The process is repeated for each subsequent codon

Termination (Bacteria)

• RF (1 or 2) mimics a tRNA and fits into A site, leading to hydrolysis of polypeptide from P site of tRNA.
• The polypeptide is released.
• RF3-GDP binds.
• The exchange of RF3-GDP for RF3-GTP causes release of RF (1 or 2).
• RF3-GTP is converted into RF3 GDP and RF3 is released

Ribosome Recycling (Bacteria)

• RRF (ribosome recycling factor) binds the A site.
• RRF looks like a tRNA ready to be translocated to P site.
• RRF recruits EF-G-GTP, the EF-G-GTP --> EF-G-GDP moves the uncharged tRNA from the P site to the E site.
• The RRF moves to the P-site.
• The Ribosome then dissociates, resulting in EF-G-GDP & RRF.
• IF3 binds the small SU to prevent the ribosome SU from reassociating.

Rescue of Stalled Ribosomes by tmRNA

• The Rescue of ribosomes that stall on broken mRNA happens via tmRNA
• tmRNA has a 5' end that folds like an Ala tRNA and is charged
• The charged tmRNA binds to stalled ribosome
• After forming a peptide bond, the ribosome continues to read codons on tmRNA
• Amino acids encoded by tmRNA are added to end of the peptide, but will mark the peptide for degradation.
• mRNAs that lack stop codons translate, in contrast, a polyA tail leading to a Poly Lys sequence.
• The truncated protein with the poly Lys tail are degraded which will recruit Ski7.
• Ski7 degrades the mRNA that lacks stop codons.

Detecting Premature Stop Codons

• Nonsense-mediated mRNA decay at a premature stop codon is caused by mRNAs splicing that leaves protein at splice sites.
• (Exon Junction Complexes).
• EJCs are removed during the first round of translation.
• The stop signal happens before the EJC which will signify a problem which will degrade the mRNA

Protein Targeting

• Secreted and membrane proteins contain amino-terminal signal sequences and are translated on ER-bound ribosomes
• Mitochondrial proteins contain amino-terminal signal sequences that are used after translation.
• Proteins are glycosylated and sorted in the ER and Golgi
• Proteins are glycosylated and sorted in the ER and Golgi
• Proteins contain nuclear localization signals and export signals

Protein Secretion (Bacteria)

• Bacteria secrete proteins using SecA, B, C
• Secreted proteins can enhance attachment to the surface of eukaryotic cells and even disrupt their ability to functions e.g., cholera toxin