Translation

• Differential gene expression regulated by the combination of different transcription factors in that cell determine cell differentiation and identity/function.
• Translation (protein synthesis): the process of converting the information present in mRNA to proteins (polypeptides).
• Translation is carried out in the ribosome.
• DNA (gene) specifies mRNA nucleotide sequence, which corresponds to the sequence of amino acids in a protein (polypeptide).
• The genetic code consists of non-overlapping triplets which are read from a fixed starting point (Francis Crick, 1961).
• tRNAs mediate the interaction of amino acids with the genetic code.
• Triplet codons- 64 codons specify 20 amino acids: 61 'sense' codons and 3 'stop' or nonsense codons.
• The genetic code is identical in all organisms, few exceptions e.g. mitochondria.
• The protein coding sequences in a gene are referred to as the 'open reading frame'. When the reading frame is changed (start is a nucleotide to the left) it is less productive as there could be more stop codons.
• For most amino acids, there is more than one codon; the genetic code is 'degenerate' (third base degeneracy). The genetic code is not ambiguous- each codon specifies only one amino acid.
• Three types of RNA directly involved with translation:

  ~Messenger RNA (protein coding; codons)

  ~Transfer RNA (anticodon and amino acid)

  ~Ribosomal RNA (components of ribosomes site of protein synthesis).
• Transfer RNA: interprets codons on DNA to amino acids. Each tRNA becomes covalently linked to one specific amino acid. Each tRNA has a triplet anticodon that recognises one or more codons in the mRNA by base pairing.
• tRNAs bring the correct amino acid to the ribosome in response to a specific coin (the amino acid is added to the growing polypeptide).
• Three functions of tRNA:

  ~It binds to an amino acid, and is then 'charged'; requires specific features in tRNA

  ~It associates with mRAN molecules through the anticodon loop.

  ~It interacts with ribosomes (requires common features).

  Amino acid attachment site always CCA.
• tRNA charging: specific enzymes couple each amino acid to its appropriate tRNA molecule. Covalent linkage of the tRNA 3' -end to the cognate (related) amino acid (specified by the anticodon). Catalysed by an aminoacyl-tRNA synthetase. One enzyme per amino acid, each recognises all isoaccepting tRNAs.
• Protein synthesis machinery recognises the anticodon of the tRNA. not the amino acid associated with that tRNA.
• How is accuracy of tRNA charging maintained?

  Controlled by the aminoacyl-tRNA syntheses. Each recognises all its isoaccepting tRNAs by contacts on the acceptor stem (one invariant discriminator base) and anticodon loop.

  Contacts aligned on one face of the tRNA.
• (2) Accuracy of tRNA charging maintained.

  ~Both amino acid and selection and tRNA selection are subject to proofreading (i.e. error-checking mechanisms)

  ~Proofreading either disfavours the forward reaction (kinetic proofreading) or reverse the catalytic reaction (chemical proofreading) if the wrong component has been selected

  ~Avoids the need for absolute accuracy (slow).
• Consequences of not tRNA charging proofreading.

  ~Sticky mutation in mice causes neurodegenerative condition.

  ~Mutation affects the accuracy of the enzyme.

  -Defect is proofreading- leads to higher mis-charging with serine.
• Decoding the mRNA to tRNA molecules match amino acids to codons. Occurs on the ribosome.

  
  

  ~31 tRNA molecules but 61 sense codons.
• Some tRNAs recognise multiple codons by the 'wobble' and modified bases that allow one tRNA to decode more than one codon.
• What is the wobble hypothesis?

  Watson-Crick base pair rules between anticodon position 1 and codon position 3 (same line from codon onto anticodon) are relaxed due to flexibility and modifications of the anticodon loop.
• Single tRNAs can recognise multiple codons.
• The ribosome:

  ~Holds mRNA and charged tRNAs in the correct positions to allow assembly of the polypeptide chain.

  ~Ribosomes can make any type of protein.
• Ribosome structure:

  ~Made from 70S (50S + 30S)

  ~In eukaryotes, the large subunit has three molecules of ribosomal RNA (rRNA) and 49 different proteins in a precise pattern.

  ~The small subunit has one rRNA and 33 proteins.
• Ribosomal subunits are held together by ionic and hydrophobic forces (not covalent bonds). When not active in translation, the subunits exist separately.

  ~70S separates into 50S and 30S by low Mg2+.
• Ribosome made up of mostly RNA and some proteins that fill the gaps.
• Role of rRNA in the ribosome.

  ~rRNA is the major structural and functional component. Not just a scaffolding for the proteins (most are nonessential).

  ~Peptide bond formation is catalysed by the 23S rRNA (i.e. a ribozyme).

  ~Subunit association and tRNA binding directly involve rRNA.

  -Specific changes in rRNA conformation are associated with these events.
• Ribosomes are a collection of proteins and rRNAs that are needed for translation.
• The ribosome structure

  ~Three sites of tRNA binding on the largest ribosomal subunit.

  ~Codon-anticodon interactions between tRNA and mRNA occur at the P and A sites.
• The sites on the ribosome:

  ~A (aminoacyl tRNA or entry) site- binds with the anticodon of charged tRNA.

  ~P (peptidyl tRNA or donor) site- where tRNA adds its amino acid to the growing peptide chain.

  ~E (exit) site- where tRNA sits before being released from the ribosome.
• Three phases to polypeptide synthesis:

  ~Initiation: mRNA binding, start codon selection, binding the first aminoacyl-tRNA.

  ~Elongation: Sequential addition of amino acids as specified by the codons in the mRNA.

  ~Termination: Release of the complexed polypeptide in response to a stop codon.
• Initiation:

  ~Nucleotide sequences in mRNA signal where to start protein synthesis.

  ~The small ribosomal subunit (3'- end of 16S rRNA- ribosome binding site) binds to its recognition sequence on mRNA.

  ~Large ribosomal subunit joins the initiation complex.

  ~In eukaryotes the small subunit binds to the 5' cap on the mRNA and moves until it reaches the start codon.

  ~Initiation factors facilitate assembly of the initiation complex.
• Initiation.

  
• Initiation in bacteria: N-formylmethionyl-tRNA enters in at the P-site.
• ~Codon recognition: (Aminoacyl-tRNA entry into the A-site).

  ~Ribosomes have a fidelity function: small subunit rRNA validates H-bonds and if they have not formed between all three base pairs, the tRNA must be an incorrect match, and it is rejected.
• Elongation:

  ~Codon recognition: The anticodon of an incoming tRNA binds to the codon at the A site.

  ~Peptide bond formation: Pro is linked to Met by peptide transferase activity of the large subunit.

  ~Elongation: Free tRNA is moved to the E site, and then released, as the ribosome shifts by one codon. so that the growing polypeptide chain moves to the P site.
• Ribosomes have a fidelity function: small subunit rRNA validates H-bonds and if they have not formed between all three base pairs, the tRNA, must be an incorrect match and it is rejected.
• Elongation is a repeating process.

  1. Codon recognition (Aminoacyl-tRNA entry into the A-site).
  2. Peptide bond formation (After peptide transfer there is a new peptidyl-tRNA in the A site and a deacylated tRNA in the P site.
  3. Translocation (simultaneously, the deactivated tRNA is ejected via the E-site the peptidyl- tRNA is moved to the P-site together with its codon and the next codon is exposed in the A-site).
• Rate of protein synthesis in bacteria: 18 amino acids/sec (error rate 1/1000-10000); in eukaryotes 3-10 amino acids/sec.