Protein Biosynthesis Steps (Translation) PDF

1/7/25 Steps in protein biosynthesis ii. Elongation Ø Begins with A site is occupied by another charged tRNA (anticodon complementary to codon after start codon on mRNA) Ø Peptide bond formed between methionine and 2nd amino acid carried by 2nd charged tRNA Ø...

1/7/25 Steps in protein biosynthesis ii. Elongation Ø Begins with A site is occupied by another charged tRNA (anticodon complementary to codon after start codon on mRNA) Ø Peptide bond formed between methionine and 2nd amino acid carried by 2nd charged tRNA Ø Ribosome moves one codon along the mRNA in 5’→3’ direction E P A Steps in protein biosynthesis Ø Methionine is detached from its tRNA that is shifted to E site before being released by ribosome Ø 2nd tRNA shifted to P site and vacant A site is occupied by another charged tRNA Ø The steps in elongation process are repeated until translation is terminated (tRNA can be activated again to add another amino acid to the polypeptide chain) E P A 1  1/7/25 Steps in protein biosynthesis Elongation Involves the addition of amino acids to the carboxyl end of the growing polypeptide chain. Involves movement of ribosomes from the 5’-end to the 3’- end of the mRNA in question. Elongation factors (EF-Tu & EF-Ts) direct the binding of appropriate tRNA to the codon in the empty ‘A’ site. Eukaryotic elongation factors are designated as eEF. Requires GTP. 2  1/7/25 Steps in protein biosynthesis Peptide bond formation is catalyzed by peptidyl transferase (a 50 S ribosomal subunit protein). Peptidyl transferase transfers the amino acid from ‘P-site’ on to the amino acid at the ‘A- site’. After peptide bond formation, ribosome advances 3 nucleotides towards the 3’-end of mRNA. The process is known as translocation which causes the release of uncharged tRNA & the movement of peptidyl tRNA into the ‘P-site’. fMet UAC 5’ ║║║ 3’ AUG UUU AAG ………. UAA Phe GTP AAA EF-Tu, EF-Ts GDP + Pi 3  1/7/25 fMet Phe UAC AAA 5’ ║║║ ║║║ 3’ AUG UUU AAG ………. UAA P A GTP GDP + Pi EF-G UAC fMet Phe AAA 5’ ║║║ 3’ AUG UUU AAG ………. UAA 4  1/7/25 Ø Methionine is detached from its tRNA that is shifted to E site before being released by ribosome Ø 2nd tRNA shifted to P site and vacant A site is occupied by another charged tRNA Ø The steps in elongation process are repeated until translation is terminated (tRNA can be activated again to add another amino acid to the polypeptide chain) Steps in protein biosynthesis iii. Termination Ø Ribosome reaches a stop codon that signals the end of translation Ø No tRNA that has a complementary anticodon to the stop codon Ø Instead, a protein release factor binds to A site Ø Large subunit advances over small subunit Ø Polypeptide detached from tRNA and starts to fold to form final shape of protein Ø RNA-ribosome complex disintegrates to release large subunit, small subunit, tRNA and mRNA 5  1/7/25 Steps in protein biosynthesis Termination Occurs when one of the three termination codons moves into the ‘A-site’. Release factor, RF-1 recognizes termination codons, UAA & UAG. Release factor, RF-2 recognizes termination codons, UGA & UAA. Release factor, RF-3 stimulates the activity of RF-1 & RF-2. Release factors cause the newly synthesized protein to be released from the ribosomal complex & dissociation of the ribosome from the mRNA. fMet Phe Lys Arg GCC 5’ ║║║ 3’ AUG UUU AAG ….. CGG UAA 6  1/7/25 RF-1, RF-2, RF-3 fMet Phe Lys Arg 5’ 3’ AUG UUU AAG ….. CGG UAA GCC Protein Synthesis: Translation 7  1/7/25 Post-translational modifications of protein Trimming: Removal of Met from amino terminus, pre- peptides (signal peptide) - in Golgi apparatus Phosphorylation: involves action of kinases & phosphatases. Glycosylation: in endoplasmic reticulum & Golgi apparatus. O-linked (Ser/ Thr), N-linked (Asn) Hydroxylation: in endoplasmic reticulum (Pro & Lys). Folding: Chaperones, disulfide isomerase, prolyl peptidyl isomerase. 8  1/7/25 Protein Synthesis: Significance of polyribosomes in translation Ø During translation, there is not only one single ribosome attached to mRNA strand but a cluster of ribosomes translating the mRNA strand simultaneously - polyribosome 9