• Translation: process of converting information stored in nucleic acid sequences into proteins

The Genetic Code

• Code is a triplet: each 3 codon in mRNA specifies 1 amino acid.

• Code is comma free: mRNA is read continuously, 3 bases at a time without skipping bases.

• Code is non-overlapping: each nucleotide is part of only one codon and is read only once.

• Code is almost universal: most codons have the same meaning in different organisms

• Code is degenerate: 18 of 20 amino acids are coded by more than one codon.

  • Met and Trp are the only exceptions.

• Code has start and stop signals: AUG codes for Met and is the usual start signal.  UAA, UAG, and UGA are stop codons and specify the the end of translation of a polypeptide.

• Wobble occurs in the tRNA anti-codon: 3rd base is less constrained and pairs less specifically.

The Wobble

• Occurs at 3’ end of codon = 5’ end of anti-codon.

• Result of arrangement of H-bonds of base pairs at the 3rd position.

• Degeneracy of the code is such that wobble always results in translation of the same amino acid.

• Complete set of codons can be read by fewer than 61 tRNAs.

tRNA

• Different tRNA molecules encoded by different genes

• tRNASer carries serine

• Common features

  • Cloverleaf structure

  • Anticodon

  • Acceptor stem for amino acid binding

• Overall ribosome shape determined by rRNA

• Discrete sites for tRNA binding and polypeptide synthesis

  • P site: peptidyl site

  • A site: aminoacyl site

  • E site: exit site

Steps to Translation

• Charging tRNA

• Initiation

• Elongation

• Termination

Charging of tRNA (aminoacylation)

• Amino acids are attached to tRNAs by aminoacyl-tRNA synthetase.

• There are 20 amminoacyl-tRNA synthases capable of “charging” all tRNAs.

• tRNAs possess specific aa-tRNA synthetase recognition sites.

• Uses energy derived from ATP hydrolysis.

• Produces a charged tRNA (aminoacyl-tRNA).

Translation

• Initiation: a functionally competent ribosome is assembled in the correct place on the mRNA and ready to start protein synthesis

• Elongation: tRNA brings the correct amino acid to the ribosome,  it is joined to the nascent polypeptide chain and the entire assembly moves to the next triplet along the mRNA

• Termination: a stop codon is reached and the entire assembly dissociates to release the newly-synthesized polypeptide.

Prokaryotic vs Eukaryotic Translation

• Prokaryotes

  • Transcription and translation occurs simultaneously

  • Shine Dalgarno sequences

  • Initiating tRNA is formyl-methionine

• Eukaryotes

  • Translation occurs in cytoplasm

  • No Shine-Dalgarno sequence;

    • Initiation factor (IF- 4F) binds to the 5’ cap on the mature mRNA

    • Eukaryote AUG codon is embedded in a short initiation sequence called the Kozak sequence.

  • Initiating tRNA is methionine

Initiation

• Binding of 30S subunit and Initiation Factors

  • IF 1: associates with 30S subunit in the A site 🡪 preventing a tRNA from entering

  • IF 3: allows for 30S subunit to bind to specific site of mRNA, checks for accuracy of binding of first aa tRNA

  • IF 2-GTP: binds to 30S P site upon which fmet-tRNA binds to IF2 and IF2 moves it to P site.  Hydrolysis of GTP occurs after 50S subunit arrives 🡪 conformational change 🡪 70S ribosome

Prokaryotic Initiation

1. Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.

2. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit).

   1. mRNA 5’-AUG-3’ start codon

   2. tRNA 3’-UAC-5’ anti-codon

3. IF3 is removed and recycled.

4. IF1 & IF2  are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA subunit.

5. Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

Elongation

• At each start

  • A site is empty

  • P site contains the peptidyl tRNA

  • E site contains an uncharged tRNA

• Requires EF-Tu, EF-Ts, EF-G

  • EF-Tu-GTP recognizes and transports aminoacyl-transfer RNAs to the A site of the ribosome.

    • EF-Tu is released from the ribosome upon hydrolysis of EF-Tu bound GTP to GDP.

    • Hydrolysis of GTP is triggered by codon-anticodon pairing at the ribosome.

  • EF-Ts reactivates EF-Tu by causing the release of GDP from EF-Tu.

  • EF-G catalyzes the tRNA/mRNA translocation

• Peptide bond formation via peptidyltransferase which is a ribozyme

  • Activity catalyzed by the 23S rRNA

  • Translocation

    • New peptidyl tRNA to P-site

    • Unchanged tRNA to E site -> EF-G-GTP required by blocking aminoacyl tRNA binding to A site and blocking Release Factors (Rfs)

    • GTP is hydrolyzed and EF-G dissociates.

Formation of the Peptide Bond

• Two aminoacyl-tRNAs positioned in the ribosome, one in the P site (5’) and another in the A site (3’).

• Bond is cleaved between amino acid and tRNA in the P site.

• Peptidyl transferase (catalytic RNA molecule - ribozyme) forms a peptide bond between the free amino acid in the P site and aminoacyl-tRNA in the A site.

• tRNA in the A site now has the growing polypeptide attached to it (peptidyl-tRNA).

Termination

1. Signaled by a stop codon (UAA, UAG, UGA).

2. Stop codons have no corresponding tRNA.

3. Release factors (RFs) bind to stop codon and assist the ribosome in terminating translation.

   1. RF1 recognizes UAA and UAG

   2. RF2 recognizes UAA and UGA

   3. RF3 stimulates termination

4. Termination events are triggered by release factors:

   1. Peptidyl transferase (same enzyme that forms peptide bond) releases polypeptide from the P site.

   2. tRNA is released.

   3. Ribosomal subunits and RF separates from mRNA