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TR056/PG1005 Lecture 18 Translation Dr. Neil Docherty My Teaching Objectives • To explain the meaning of the genetic code • To introduce tRNA structure and describe the mechanism of amino acid (AA) charging • To describe the function of the ribosome in translational initiation, elongation and termination Genetic Information Directs Protein Synthesis From Messenger RNA 5’ Gene code (DNA) 3’ 3’ 5’ RNA polymerase II Intermediate (mRNA) 5’ TRANSCRIPTION 3’ NUCLEUS CYTOPLASM ribosome (+rRNAs) membrane rER-Golgi TRANSLATION /TARGETTING organelle secretory vesicle tRNA (+ amino acid) free ribosomes cytoplasmic protein DNA to mRNA Transcription Summary • Chromatin remodelling (e.g. acetylation) • TATA box binding • Initiation complex assembly/RNA polymerase II recruitment • 5’ to 3’ synthesis and 5’ G-capping • Termination and 3’ poly-A tailing • Intron removal (splicing) • Nuclear export RNA to Protein Paradigm 5’ Mature mRNA 3’ Triplet codes (codons) Complimentary coding tRNA-AAs (anti-codons) CODON-ANTI-CODON MATCHING AA1 - AA2 PEPTIDE BOND CATALYSIS Covalent peptide bond ON RIBOSOMES The Genetic Code • Triplet codes (codons) specify particular amino acids • Due to a complimentary anticodon found on tRNA charged with specific amino acids N.B. • Redundancy • Start and Stop Codons Redundancy most frequent at third base (relevant later) Reading Frames and The Start Codon 5’ Mature mRNA 3’ Mature mRNA 3’ AUG GUA C Met Val 5’ AUG GUA C Trp Tyr In principle, 3 reading frames exist However, AUG-Met act as a start signal and sets reading frame (It is located by the ribosomal machinery at start of translation, Hence N-terminal AA always methionine) Transfer RNA (tRNA) CODON- ANTICODON-AA mRNA tRNA (80nts post-splicing) Unusual modified Bases e.g. pseudouridine Wobble Base Pairing (tRNA) • Several mRNA codons can code for a specific AA • Change is mainly due to differences at 3rd “wobble” position • The third base of the anticodon on tRNA is frequently a modified base • Modified bases have multiple binding capacities • Hence redundancy in codons often a result third base wobble pairing between codon and anticodon Charging of tRNA with AA Two Steps catalysed by AMINOACYL tRNA SYNTHETASES; 1) ATP dependent adenylation of AA 2) Ester linkage of AA to 3’ OH of ribose sugar of 3’ tRNA nucleotide Aminoacyl tRNA Synthetases • Each AA has its own synthetase (Hence, n=20) • e.g. One synthetase exists for attachment of tryptophan to a tRNA, the anti-codon of which is complimentary to the codon sequence for tryptophan specified in the genetic code Editing Features of Synthetase Enzymes Ensure Fidelity of tRNA-AA Matching What mechanisms allow synthetases to accurately match AA-tRNA pairs (1:40,000 error rate)? 1) The correct AA has a high affinity for AA binding pocket of enzyme (too large=excluded) 2) The correct tRNA is associated with the enzyme by fit of anti-codon sequences into 3 adjacent nucleotide binding pockets 3) Adenylated AA is shunted to a new site following tRNA binding. If it fits, it is hydrolysed (=removed) The Ribosome • Two subunit complex translational machinery composed of Proteins 1/3 and rRNA (2/3). Charged with the following tasks; -Matching anti-codons and codons (SMALL SUBUNIT) -Catalysing peptide bond formation (LARGE SUBUNIT) LARGE SMALL Initiation of Translation Recall that Met is always the first AA of a nascent polypeptide • A specific tRNA called the initiator is charged with Met • Initiator is bound to the small ribosomal subunit in association eukaryotic initiation factor proteins (EIFs) • Binding of 5’ cap of mRNA proceeds • 5’ to 3’ searching of mRNA for first AUG codon • EIF disassociation, large subunit binding • TRANSLATION CAN BEGIN Ribosomal Binding Sites Four binding sites important 1) mRNA 2) tRNA 1=A (aminoacyl tRNA site) 3) tRNA 2=P (Peptidyl tRNA site) 4) tRNA 3=E (tRNA exit site) Elongation After step 3 a new aminoacyl tRNA is free to bind to A site Obviously 3’ downstream! Requires; 1) Elongation Factors 2) GTP hydrolysis Catalytic Mechanism Function of large subunit 23S rRNA driven (ribozyme) 1) Proton abstraction from amino nitrogen of A site AA 2) Reaction of P site AA carboxyl group with A site amino nitrogen 1) Adenine protonation of P site tRNA to release hydroxylated tRNA 2) Addition of one new peptide bonded AA to nascent protein Termination Translation terminates at STOP CODONS UAA UAG UGA No corresponding tRNA Release Factors bind H2O added to C-terminal end Frees chain mRNA release Ribosomal subunit disassociation Your Learning From Today Should focus on being able to; 1) Appreciate and explain how nucleotide triplets in mature mRNA specify a code for amino acids 2) Explain the concept and basis of redundancy in the genetic Code 3) Describe the structure and function of the ribosome.