M2.4: DNA Translation

Types of Ribosomes

• PROKARYOTES: Monosome 70s

  • large subunit: 50s; small subunit: 30s

• EUKARYOTES

  • large subunit: 60s; small subunit: 40s

Ribosome: important sites

• mRNA binding site

  • found in small subunit of ribosome

• Aminoacyl or Acceptor (A) site: binding site of tRNA + amino acids

• Peptidoglycan or Peptide (P) site: tRNA + growing polypeptide

• Exit site (E): tRNAs that no longer have a bound amino acid exit from the ribosome

• Catalytic site: covalent bond (peptide bond) between 2 amino acids

  • bond forms between C and N of the 2 aa

Peptide bond synthesis

carbonyl end of the 1st amino acid and the amino end of the 2nd amino acid are connected

Translation stages

1. initiation

2. elongation

3. termination

Shine dalgarno sequence

ribosomal binding site that serves as an initiating signal for protein synthesis

• enables initiation of protein synthesis by aligning the ribosome with the start codon

• indicates where protein synthesis starts, thus ensuring that genes are read correctly

• found between untranslated region and sequence coding for the 1st aa (which explains why proteins are generally shorter than mRNA: since shine dalgarno sequence is not translated)

Wobble concept

• codons don’t follow specific base pairing

• no need for 1:1 correspondence between codons and tRNA (1 tRNA can recognize multiple codons which code for the same sequence)

Initiation (EUKARYOTES)

• ribosome, mRNA, and initiator tRNA contains the 1st aa in the protein (usually Met) come tgt to form the initiation complex

• tRNA containing Met attaches to the small ribosomal subunit. tgt, they bind to the 5’ end of the mRNA by recognizing the 5’ GTP cap (added during processing in the nucleus)

• afterwards, tRNA + small ribosomal subunit go down the mRNA in the 3’ direction, stopping when they reach the start codon (AUG)

Initiation (PROKARYOTES)

• small ribosomal subunit attaches to the shine-dalgarno sequences (which is just before the start codons)

Elongation

• Met-carrying tRNA starts in the P site (middle slot of the ribosome). next to it, a fresh codon is exposed in the A site

  • A site will be the “landing site” for the next tRNA, one whose anticodon is a complementary match for the exposed codon

• once the complementary tRNA has landed in the A site, a peptide bonds will form between the amino acids and connect them to each other (Met will be transferred frm the 1st tRNA to the aa of the 2nd tRNA in the A site)

  • Met forms the N-TERMINUS of the polypeptide (end of the polypeptide with an exposed amino group)

  • other aa is the C-TERMINUS (end of the polypeptide with an exposed carboxyl group); new aa are progressively added to the C-terminus

• once a peptide bond is formed, the mRNA is pulled onward through the ribosome by 1 codon; this shift allows the 1st empty tRNA to drift out via the E site and also exposes a new codon in the A site, thus allowing the cycle to repeat

Termination

• stop codons are recognized by release factors (proteins which fit into the P site)

  • release factors disrupt the enzyme that normally forms peptide bonds (they make it add a H2O molecule to the last aa of the chain, which separates the chain from the tRNA and releases the newly-synthesized protein)

• after the small and large ribosomal subunits separate from the mRNA and from each other, each element can take part in another round of translation

Eukaryotic translation

• translation is separated spatially and temporally from transcription (since sites for transcription and translation are separated by organelles)

• larger ribosomes

• longer rRNA (expansion segments needed for ribosome assembly and translation regulation and specificity)

  • expansion segments are not coded by DNA/not part of the gene

• no shine-dalgarno sequence

  • instead use cap-dependent translation

• initiation starts with the small subunit associating with m^7 G cap (CAP-DEPENDENT TRANSLATION)

• eukaryotic initiation factors (eIFs), eukaryotic elongation factors (eFFs), eukaryotic release factors (eRFs)

• tRNAiMet: unformylated Met (which is why first amino acid is always fMet)

• presence of Kozak sequence (A/GNNAUGG): increased efficiency of translation initiation

  • has purine base (adenine/guanine); then any other base

  • enhances efficiency of translation initiation

• eukaryotic mRNAs (10 hrs) are longer lived than bacterial mRNAs (5 mins)

  • can live longer without being degraded)

• Poly-A binding proteins are required to form the initiation complex

closed-loop translation

• avoids wasting energy in translating partially degraded mRNA

• efficient ribosome recycling