FOB 10 translation

• how is mRNA translated


  4 letters into 20 amino acids, many more possible combinations. multiple codes for one amino acids. tRNA (transfer rna) has an adaptor with a anti-codon, the reverse compliment and the relevant amino acid attached.
• codon


  each amino acid is encoded by a triplet of nucleotides, a codon. translation occurs through the successive reading of these triplets in a non-overlapping fashion. the starting position of the first codon determines the reading frame and hence the amino acid sequence. in general, there are 3 possible reading frames for any one mRNA sequence. however, normally the reading frame is defined by the start of a codon AUG (methionine) which allows the same amino acid sequence to be produced every time
• important codon codes


  aug start, uaa, uag, uga are stops. degeneracy code: the alternate codes for a amino acid. aug in prokaryotes is called formylethionine
• tRNA


  single-stranded RNA molecule 73-93 nucleotides held by complimentary hairpin regions. two-dimensional image shows a clover leaf like structure with 4 major arms (D arm and T(trident)C arm recognition, interact with ribosomal structure and other tRNa, other is anticodon which binds in opposite directions) 3d structure looks like L. larger tRNAs have a 'extra' arm. opposite anticodon contains the acceptor stem which binds an amino acid to the free 2' or 3' hydroxyl group forming aminoacyl group
• wobble


  if each tRNA only recognised one codon, cells would need 61 tRNA, but cells have 31< >45. the first nucleotide in anti codon C or A usually follow the rules, but U and G can bind with A or G and C and U respectively. Iosinate is modified A (everything but G). when the first base of the anti-codon s C or A, only 1 codon is recognised. when U or G, two codons are recognised (AG or CU). when I is first 3 nucleotides are recognised AUC.
• ribsomes


  composed or rRNA 65% (enzymatic) and proteins 35% (structure). activated tRNA (amino acyl tRNA) molecules and mRNA. made of upper and lower subunit. pro: 50s + 30s total 70s. E 60s + 40s total 80s. S = sediment coefficient, relates to the molecular weight, volume and diameter of the molecule. ribosomal RNA forms structural core and proteins act as secondary element. no protein in active site rRNA is ribozyme. 2 subunits come together to form a cleft that mRNA passes through as the ribosome moves
• 5 stages of protein synthesis

  activation of amino acids, initiation, elongation, termination + release, folding and post-translational processing.
• activation of amino acid


  attachment of aa to tRNA aminoacyl-tRNA. components: 20 amino acids, 20 aminoacyl-tRNA synthetases (add amino acid to tRNA), 32+ tRNAs, ATP, Mg2+. aminoacyl-tRNA synthetases esterify the amino acids onto their corresponding tRNA creating an aminoacyl-tRNA, same synthase for amino acids with more than one tRNA. two classes of synthases that only differ in how they enzymatically attach the amino acid.
• initiation


  mRNA binds to lower ribosomal subunit followed by the initiating aminoacyl-tRNA (fMet or Met) binding to the start codon (AUG). finally, the larger ribosomal subunit binds to form the initiation complex
• elongation


  newly forming polypeptide chain is lengthened by continuous binding of further tRNA molecules to their corresponding codons on the mRNA sequence within the ribosomal complex
• termination


  completion of the new polypeptide chain is signalled by a termination codon (UAA, UGA and UAG) peptide is released and ribosomal complex is recycled for another round of synthesis.
• folding and post translational modifications


  new polypeptide chain folded into its three-dimensional shape. may also undergo modifications (trimming of peptide chain, acetylation and phosphorylation)
• activating aminoacyl tRNA


  class 1 and 2. aminoacyl TRNA synthase turns amino acid into aminoacyl. two phosphates are trimmed off nucleotide and this energy attaches amino acid to a phosphate through ester linkage. activates carboxylic acid group allowing nucleophilic attack to attach to tRNA. 2: brings aminoacyl-AMP in contact with 3' free hydroxyl group which undertakes a nucleophilic attack on the carboxylic acid group. 1: aminoacyl amp is linked to carbon 2 and then undergoes nucleophilic attack then transesterification links it to carbon 3. covalently linked to tRNA with ester bond.
• aminoacyl tRNA proofreading


  only correct amino acid fits within the active site, trna has recognition sequences to ensure trna correct, proofreading ability to remove the wrong amino acid from trna break it apart
• initiation of synthesis


  ribosome has E,P, and A sites. A- binds incoming aminoacyl-tRNA. P - contains the extending peptide chain attached to a tRNA. E - binds uncharged tRNAs that have finished/are exiting. small subunit binds mRNA, guided into place by the shine-Dalgarno sequence (prokaryotes), places start codon on P site. initiation factor 1 binds preventing new trna from entering A site, iF 3 binds on E preventing large subunit from binding. tRNA start codon fMET with its own specific tRNA bind. then large subunit is added ontop and ignition factors break off. Euk more initiation factors
• elongation (bacteria)


  requires: ribosomal initiation complex, aminoacyl-tRNAs, elongation factors (EF-Tu, Ts, G) and GTP. 3 steps: next tRNA binds to GTP bound EF-Tu then A site of subunit, hydrolyzing GTP. Ts recharges Tu with GTP. Form peptide bond w 2 aa, done by peptidyle transferase 23S rRNA ribosome. aas are attached to tRNAs in site A n P. transfer of aa group in site P to second aa group in A. Translocation of ribosome. moves from one codon towards 3' of mRNA. shifts now empty tRNA from P to E and peptide chain from A to P. requires hydrolysis of GTP attached to EF-G
• termination


  uaa, uag, uga enter site A causing binding of termintaion/release factors 1,2,3. causing hydrolysis of terminal peptidyl-tRNA bond, release of free polypeptide and last tRNA. dissociation of the 70s ribosome into corresponding subunits
• building polypeptide chain


  amino acids link from P to A site and chain builds on P trna. amine/peptide linkage when amino group and carboxylic acid group are brought into close proximity. ester bond to trna activates, and breaks as soon as amine bond forms.
• eukaryote vs prokaryote translation and transcription


  prokaryotes have no nucleus therefore mRNA does not have to be exported out of the nucleus, ribosomes can synthesise proteins immediately
• post-translational modifications


  to assume native conformation and become functional. amino and carboxyl-terminal modifications: removal of Fmet from N-terminal, 50% eukaryotes are acetylated on N terminal. loss of signal sequences: sequence used to guide proteins to correct cellular location. modification of aa's: phosphorylation, acetylation, methylation. attachment of carbohydrate side chains (glycoproteins) or other macromolecules (lipoproteins), addition of prosthetic groups, proteolytic cleavage (activation), formation of disulphide bonds