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Protein Synthesis (Translation) Proteins Synthesis Translation Process by which ribosomes convert the information carried by mRNA to synthesize new proteins mRNA is translated from 5 to 3-end producing a protein (peptide) synthesized from amino to carboxyl end Requirements 1. Ribosomes 2. Amino acids 3. mRNA 4. tRNA 5. Enzymes 6. Protein factors (initiation, elongation & release factors) 7. ATP & GTP as source of energy Genetic Code CODON: a sequence of three adjacent nucleotides (bases) One or more sequences (codons) for each amino acid (61 for 20 amino acids) If there are more than one codon for one amino acid then these codons are known as synonyms Starter / initiator codon (always AUG for methionine) Stop codons or termination codons (UAA, UAG & UGA) Genetic Code (64 Possible codon sequences) Phe UUU, UUC Leu CUU, CUC, CUA, CUG, UUA, UUG ILeu AAU, AUC, AUA Met AUG Val GUU, GUC, GUA, GUG Ser UCU, UCC, UCA, UCG, AGU, AGC Pro CCU, CCC, CCA, CCG Thr ACU, ACC, ACA, ACG Ala GCU, GCC, GCA, GCG Tyr UAU, UAC If 4 codons for one amino acid; most of these codons differ in 3rd nucleotide Genetic Code (64 Possible codon sequences) His CAU, CAC Gln CAA, CAG Asn AAU, AAC Lys AAA, AAG Asp GAU, GAC Glu GAA, GAG Cys UGU, UGC Trp UGG Arg CGU, CGC, CGA, CGG, AGA, AGG Gly GGU, GGC, GGA, GGG Proteins Synthesis (Steps) I. Activation of amino acids (formation of amino-acyl-tRNAs or charged tRNA) Amino-acyl-tRNA-synthetases recognize and catalyze the covalent attachment of a specific amino acid to CCA arm of tRNA Amino-acyl-tRNA Amino acid + tRNA ATP AMP + PPi 2Pi First amino acid is always methionine (met-tRNA) II. Codon-anticodon recognition Codon (on mRNA) & anti-codon (on tRNA) are anti-parallel to one-another Proteins Synthesis (Steps) III. Initiation Proper mRNA is selected by a ribosome ; ribosome attaches to it and reads the message from 5 to 3 end of mRNA Ribosome dissociates into 40s & 60s sub-units Formation of Pre-initiation complex (PIC) [40 s rRNA + met-tRNA + GTP + initiation factors (eIFs)] mRNA binds to PIC forming 43 s initiation complex Proteins Synthesis (Steps) d. Binding of 43s (IC) with 60s ribosome forms 80s initiation complex, this complex contains P, A & E sites (“P” for peptidyl tRNA , “A” for new amino-acyl-tRNA & “E” for exit) Starter / initiator codon (AUG) is identified by tRNA near the 5 end (cap end) of mRNA This is determined by other sequences ,“Kozak consensus sequences” that surround “AUG” met-tRNA occupies “P” site and is ready for elongation Elongation A cyclic process; one amino acid is added at a time Sequence determined by the order of codons in mRNA Requires “Elongation factors” (EFs) Steps a. Binding of amino acyl-tRNA to the “A” site b. Peptide bond formation of this incoming amino acid with the carboxyl group (end) of met-tRNA (or growing peptide chain) at “P” site Catalyzed by “Peptidyl-transferase” – a component of 28s rRNA (ribozyme) of the 60s ribosomal subunit Elongation c. This results in attachment of growing peptide to tRNA in the “A” site d. Translocation – Now the ribosome advances three nucleotides (1 codon) towards 3-end of mRNA; results in Movement of free tRNA (uncharged) from “P” to “E” site for release EF2 displaces peptidyl-tRNA from “A” to “P” site “A” site is now open for another amino-acyl-tRNA for another cycle of elongation Termination Elongation continues until a termination codon is encountered Termination occurs when one of the three termination codons moves into “A” site These codons are recognized by release factors Release factors bind to and induce peptidyl transferase to release the polypeptide from tRNA Post-translational modifications Trimming Covalent modifications Phosphorylation Glycosylation Hydroxylation Other covalent modifications Formation of disulfide links Addition of carboxyl, acetyl group etc. Addition of prosthetic group