BIOL10221 Molecular Biology Lecture 2 PDF

BIOL10221 “Molecular Biology” Dr David Boam Module 7 – From RNA to Protein Lecture 2: The ribosome and  Aims of this lecture to introduce the basic mechanism of translation How mRNA is used to code for a polypeptide chain Stages in translation The role of cofac...

BIOL10221 “Molecular Biology” Dr David Boam Module 7 – From RNA to Protein Lecture 2: The ribosome and  Aims of this lecture to introduce the basic mechanism of translation How mRNA is used to code for a polypeptide chain Stages in translation The role of cofactors and nucleotides in translation The role of the ribosome in translation  Protein synthesis Machinery (ribosomes, cofactors) Energy – (ATP and GTP) Mechanism – Three stages: Initiation Elongation Termination  The Ribosome RNA/protein complex composed of two subunits Large subunit Small subunit Large subunit Small subunit  Why 2+2 ≠ 4 (S)Svedberg - a measure of the sedimentation rate of suspended particles centrifuged under standard conditions – shape and size. rRNA’s have elaborate secondary structures made up of 40 or more stem loops 16S rRNA  Ribosome structure The ribosome illustration shows the position of a tRNA in the A-site 30S subunit of E. coli, showing examples of within the 70S ribosome in its 16S rRNA helices from a biochemically pre-translocational state derived model Reproduced from Cell, 1997, v. 88, http://www.bio.ic.ac.uk/research/vanheel/ pp. 19-28 vanheel.html http://www.bio.ic.ac.uk/research/ vanheel/vanheel.html  Amino acid activation H O Attachment of amino H2N C C OH OH acids to tRNA 3’ R ATP “acceptor arm” 2 step process H O H2N C C R O P ribose adenine AA + ATP AA-AMP P ribose adenine H O + PP H2N C C R O · AA-AMP + tRNA AA- tRNA + AMP  Initiation – finding the right codon Shine - Dalgarno sequence (prokaryotes) (aka Kozak sequence in eukaryotes) Conserved. Interacts with 16S rRNA of the 30S small subunit to identify the site of initiation of protein synthesis Two tRNA’s for methionine, one for initiation (tRNAi), one for elongation. Prokaryotic initiating met is formylated (fmet)  Initiation of translation Active IF2 GTP Meti P A large subunit joins Initiator tRNA to complete (formylmethionyl) initiation complex. and mRNA join complex ATPADP IF1 IF3 GTP GDP Initiation inactive IF2 factors/GTP bind to GDP 30S subunit 5' mRNA 1 2 3'  Initiation factors IF1 – Blocks A site to tRNAi-met, inhibits premature 30S-50S interaction IF2 – tags tRNAi and regulates entry into ribosome IF3 – – Inhibits premature 30S-50S interaction – Stabilises free 30S – Accuracy check for tRNAi-met binding  ternary complex AA2 Elongation GTP Tu peptide bond GTP G formation Met Met Met i Met AA2i AA2i AA2i 5' mRNA 1 2 3' 5' 1 2 3 3' 1 2 3 3' 1 2 3 4 3' P A P A P A P A P site - peptidyl- Tu GDP G GDP tRNA site, Where peptide bonds A site - form tRNA-Met i is the aminoacyl-tRNA only tRNA which site - where can bind to the P incoming aminoacyl-tRNA site Association of EF-G-GTP and binds Elongation factors ejection of empty tRNA from the P ternary complex = necessary site aminoacyl tRNA-EFTu-GTP Ribosome translocates so peptidyl tRNA is now in P site, A site is free Binding to ribosome A site  Elongation factors EF-Tu – mediates aminoacyl-tRNA entry to ribosome EF-G – mediates translocation Elongation animation  Termination aa98 RF aa99 GTP aa100 i Three prokaryotic GDP release factors – RF1 – UAA/UAG 98 99 100 T 3' RF2 – UAA/UGA RF3 – helps RF1 or 2 P A bind to ribosome - GTPase RF-GTP binds to A Hydrolysis of Dissociation site when termination codon polypeptide of tRNA and appears chain from RF tRNA Summary of translation (animation)  The role of GTP GTP - Guanosine triphosphate Provides energy (hydrolysis) GTP GDP + Pi When is GTP involved in protein synthesis? Initiation - addition of large subunit Elongation - addition of aminoacyl-tRNA by EF-Tu Peptide synthesis and translocation Termination - release of peptide chain and dissociation of ribosome Protein synthesis -summary Requires: – Ribosomes – tRNA – mRNA – accessory factors – Energy (ATP and GTP) Three stages: – Initiation (initiation codon identification, assembly) – Elongation (polymerisation, translocation, energy driven) – Termination (energy driven)  ILOs of this lecture You should understand – Initiation elongation and termination of translation – The role of the ribosome in translation and in translation start site selection – The identity and roles of translation cofactors – The roles of nucleotide triphosphates in translation