Protein Biosynthesis - Translation PDF

1/7/25 70 s 50 s 30 s - 5 s RNA 34 proteins 23 s RNA 16 s RNA 21 proteins Prokaryotic ribosome 80 s 60 s...

1/7/25 70 s 50 s 30 s - 5 s RNA 34 proteins 23 s RNA 16 s RNA 21 proteins Prokaryotic ribosome 80 s 60 s 40 s 5 s RNA 45 proteins 28 s RNA 18 s RNA 33 proteins 5.8 s RNA Eukaryotic ribosome 1  1/7/25 PROTEIN BIOSYNTHESIS - TRANSLATION ii. Ribosomal RNA (rRNA) Ø Three sites:- - mRNA-binding site (on small sub-unit) - E site (exit site for uncharged tRNA before it is let go by the ribosome) - P site (peptidyl-tRNA binding site) - A site (aminoacyl-tRNA binding site) PROTEIN BIOSYNTHESIS - TRANSLATION iii. Transfer RNA (tRNA) Ø tRNA transports a specific amino acid from cytoplasm to ribosomes Ø Single-stranded molecule, folds to form a ‘clover-leaf’ shape due to base-pairing Ø Has 3 loops formed by regions where bases are not paired 2  1/7/25 PROTEIN BIOSYNTHESIS - TRANSLATION iii. Transfer RNA (tRNA) Ø Anticodon loop – contains a sequence of 3 nucleotides that complements the codon on mRNA Ø The 3’ end is the amino acid attachment site where specific amino acid is attached Base sequence of CCA-3,-OH Aminoacylation / charging = process of binding amino acid → charged tRNA (catalyzed by aminoacyl-tRNA synthetase) Ø Because of their ability to both carry a specific amino acid & to recognize the specific codon on mRNA, tRNA is known as adaptor molecule. PROTEIN BIOSYNTHESIS - TRANSLATION 3  1/7/25 PROTEIN BIOSYNTHESIS - TRANSLATION Aminoacyl tRNA synthetases ž Enzyme recognizes both a specific amino acid & the tRNA, corresponding to that amino acid. Activation of each type of amino acid requires a specific enzyme of this family. Each enzyme catalyzes a two step reaction resulting in the covalent attachment of an amino acid to the 3’ end of its corresponding tRNA. Codon recognition by tRNA ž Recognition of a particular codon on an mRNA sequence is accomplished by the anticodon sequence of tRNA. Binding of tRNA anticodon to the mRNA codon follows the rules of complimentary, antiparallel binding (mRNA codon is “read” 5’→3’ by an anticodon pairing in the “flipped” 3’→5’ orientation) When writing the sequences of both codon & anticodon, the nucleotide sequence must ALWAYS be listed in the 5’→3’ order. 4  1/7/25 PROTEIN BIOSYNTHESIS - TRANSLATION Ø Three stages:- i. Initiation ii. Elongation iii. Termination PROTEIN BIOSYNTHESIS - TRANSLATION Ø Three stages:- i. Initiation ii. Elongation iii. Termination i. Initiation Ø Small subunit of ribosome binds to the 5’ end of mRNA – slide along mRNA until reaches start codon Ø Charged tRNA with anticodon UAC (met-tRNA) binds to small subunit of ribosome Ø Large subunit binds to small subunit – first tRNA occupies P site and A site available for the next tRNA. Ø Shine Dalgarno sequence 5  1/7/25 Steps in protein biosynthesis Pathway of protein biosynthesis is called as ‘translation’ as the language of the nucleotide sequence on the mRNA is translated into the language of amino acid sequence. The mRNA is translated from its 5’-end to its 3’-end. The protein is synthesized from its amino terminal to its carboxyl terminal end. Prokaryotic mRNAs have several coding regions (each has its own initiation sequence) & produces a separate species of polypeptides. Steps in protein biosynthesis Each eukaryotic mRNA codes only for one polypeptide chain. There are three separate steps in translation process (initiation, elongation & termination). The produced polypeptide may be further modified by “Post- translational modifications”. Eukaryotic protein biosynthesis resembles that of prokaryotic protein synthesis in most details. 6  1/7/25 Steps in protein biosynthesis Initiation involves the assembly of components of the translational system. Components include two ribosomal subunits, mRNA, aminoacyl tRNA, GTP & initiation factors. In prokaryotes, three initiation factors (IF-1, IF-2 & IF-3) are known whereas in eukaryotes at least eight initiation factors designated as eIF are present. 7  1/7/25 Steps in protein biosynthesis Two mechanisms by which the ribosome recognizes nucleotide sequence of mRNA that initiate translation. Mechanism 1 q In E.coli, nucleotide sequence (5’-AGGAGG-3’) known as Shine Dalgarno sequence is located 4 to 7 bases before the AUG codon on mRNA. q The 16 S rRNA of 30 S ribosomal subunit has a nucleotide sequence that is complimentary to all or part of the Shine Dalgarno sequence. q Therefore, mRNA 5’-end & 16 S rRNA can form complimentary base pairs, thus facilitates binding & positioning of mRNA on the 30 S ribosomal subunit. 16 S rRNA 30 S ribosomal subunit UCC UCC ║║║ ║║║ AGG AGG mRNA 5’-end 3’-end Shine Dalgarno sequence 8  1/7/25 Steps in protein biosynthesis Mechanism 2 AUG codon in the beginning of the message is recognized by a special initiator tRNA. In bacteria, this tRNA carries N-formylated methionine. In eukaryotes, it carries methionine. In both prokaryotic & eukaryotic cells, N-terminal methionine may be removed. Initiation factors aid in the formation of 30 S initiation complex. GTP is cleaved & initiation factors are released when 50 S subunit arrives to form 70 S initiation complex. fMet 3’ 5’ UAC 5’ ║║║ 3’ AUG UUU AAG ………. UAA 30 S IF-1 IF-3 IF-2 50 S GTP IF-1, IF-2, IF-3 GDP + Pi 9  1/7/25 Inititation complex Initiation factors 1, 2, 3, f-met, 30S, 50S, GTP, start codon AUG, mRNA, Shine Dalgarno sequence. Inititation complex Base pairing between the 16s RNA (part of the ribosome) with Shine Dalgarno sequence. 10