NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY UNIT 4: TRANSLATION Introduction to Genetic code: Elucidation of genetic code, Codon degeneracy, Wobble hypothesis and its importance, Prokaryotic and eukaryotic ribosomes. Components of translation. Activation of tRNA. Mechanism of translation: Initiation, Elongation and Termination of protein synthesis, Differences between prokaryotic and eukaryotic protein synthesis. Post-translational modifications and its importance. Protein splicing. Protein targeting: signal hypothesis and cotranslational processing, transportation. Inhibitors of protein synthesis. The Genetic Code Elucidating the Genetic Code • A triplet code is required: 43 = 64, but 42 = 16 - not enough for 20 amino acids • But is the code overlapping? • And is the code punctuated? The Nature of the Genetic Code • A group of three bases codes for one amino acid • The code is not overlapping • The base sequence is read from a fixed starting point, with no punctuation • The code is degenerate (in most cases, each amino acid can be designated by any of several triplets Assignment of "codons" to their respective amino acids was achieved by in vitro biochemistry • Marshall Nirenberg and Heinrich Matthaei showed that poly-U produced polyphenylalanine in a cell-free solution from E. coli • Poly-A gave polylysine • Poly-C gave polyproline • Poly-G gave polyglycine • But what of others? Features of the Genetic Code • All the codons have meaning: 61 specify amino acids, and the other 3 are "nonsense" or "stop" codons • The code is unambiguous - only one amino acid is indicated by each of the 61 codons • The code is degenerate - except for Trp and Met, each amino acid is coded by two or more codons • Codons representing the same or similar amino acids are similar in sequence • 2nd base pyrimidine: usually nonpolar amino acid ,2nd base purine: usually polar or charged aa . R.S.UPENDRA RAJU Page 1 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY • Codon – specifies the sequence of amino acids, Initiation (start) codon--- AUG – methionine • Every protein in a cell starts with methionine ,Termination (stop) codons--UAA, UGA, UAG. • Genetic code is Universal,Degenerate - some amino acids are specified by more than one codon • 64 possible codons and only 20 amino acids Third-Base Degeneracy Codon-anticodon pairing is the crucial feature of the "reading of the code" • But what accounts for "degeneracy": are there 61 different anticodons, or can you get by with fewer than 61, due to lack of specificity at the third position? • Crick's Wobble Hypothesis argues for the second possibility - the first base of the anticodon (which matches the 3rd base of the codon) is referred to as the "wobble position" The Wobble Hypothesis • The first two bases of the codon make normal (canonical) H-bond pairs with the 2nd and 3rd bases of the anticodon • At the remaining position, less stringent rules apply and non-canonical pairing may occur • The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A) • Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too. R.S.UPENDRA RAJU Page 2 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY Prokaryotic vs Eukaryotic Ribosomes Prokaryotic Ribosomes Generally prokaryotic ribosomes are called 70S ribosomes, which are smaller than eukaryotic ribosomes (Taylor, 1998). Ribosomes consist of two subunits, and these two subunits are called 30S and 50S, the smaller unit and the larger unit respectively. These ribosomes units are denoted by Svedberg (S) values depending on the rate of the sedimentation in the centrifugation . In prokaryotes, rRNA is organized into three strands in ribosomes . Eukaryotic Ribosomes Smaller subunit and larger subunit of eukaryotic ribosomes are described as 40S and 60S respectively, and the whole ribosome is 80S. This is lager than the prokaryotic ribosome. The rRNA in ribosomes has four strands. Ribosomes are produced in the nucleolus, in a special position in nucleus. Ribosome Type Eukaryotic Prokaryotic Sedimentation 80 S 70 S coefficient 6 6 Molecular mass ~3.2×10 Da ~2.0×10 Da Diameter ~250-300 Å ~200 Å Large Sedimentation 60 S 50 S subunit coefficient Molecular mass ~2.0×106 Da ~1.3×106 Da Proteins 47 33 rRNAs 28 S rRNA (3354 nucleotides) 23S rRNA (2839 nucleotides) 5 S rRNA (120 nucleotides) 5S rRNA (122 nucleotides) 5.8 S rRNA (154 nucleotides) Small subunit Sedimentation 40 S 30 S coefficient Molecular mass ~1.2×106 Da ~0.7×106 Da Proteins 32 20 rRNAs 18S rRNA (1753 nucleotides) 16S rRNA (1504 nucleotides) Translation Process of converting information stored in nucleic acid sequences into proteins Sequences of mRNA (messenger RNA) are translated into unique sequence of amino acids in a polypeptide chain. Translation takes place in the cytoplasm, Exception are few proteins coded by mitochondrial and chloroplastic DNA Translation is Performed on ribosomes Components of translation process R.S.UPENDRA RAJU Page 3 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY 1. Template – mRNA.2. Genetic code.3. tRNAs (transfer RNAs) Linked to amino acids 4. Ribosomes both large and smaller sub units. 5. Many accessory proteins.( translation factors) 6. Some energy (GTP hydrolysis) 1.Template – mRNA m RNA is a Single stranded molecule of RNA that encodes sequence of the polypeptide Transcribed and processed in the nucleus and then exported into cytoplasm 5’ end has binding sites for translation initiation, Middle is a coding sequence, 3’ end regulates stability of mRNA Prokaryotic and eukaryotic mRNAs Both prokaryotic and eukaryotic mRNAs contain untranslated regions (UTRs) at their 5´ and 3´ ends. Eukaryotic mRNAs also contain 5´ 7-methylguanosine (m7G) caps and 3´ poly-A tail. Prokaryotic mRNAs are frequently polycistronic: They encode multiple proteins, each of which is translated from an independent start site. Eukaryotic mRNAs are usually monocistronic, encoding only a single protein. 2.Genetic code Codon – specifies the sequence of amino acids. Initiation (start) codon--- AUG – methionine ,Every protein in a cell starts with methionine .Termination (stop) codons--UAA, UGA, UAG. Genetic code is Universal. Degenerate - some amino acids are specified by more than one codon 64 possible codons and only 20 amino acids R.S.UPENDRA RAJU Page 4 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY 3.tRNA t RNA is a L-shaped secondary structure which Deliver amino acids to the translational complex It Serves as adapters between codons in mRNA and amino acid. Mainly Consists 4 stems and 3 loops in its structure. Anticodon loop consists decoding triplet - localized on the anticodon stem.Anticodon and amino acid are at the opposite arm of the L 4.Ribosome- Molecular machines that coordinate the interplay of charged tRNA, mRNA and proteins that lead to proten synthesis. Ribosomes can be dissociated into – Large (50S)-23S (peptide bond formation)- 5S – Small (30S)-16S (pairs with Shine-Dalgarno sequence on mRNA R.S.UPENDRA RAJU Page 5 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY Protein synthesis or translation stages & essential components Activation of amino acids It is the linkage of amino acid to its tRNA. It is a crucial step because the attachment of a given amino acid to a particular tRNA establishes the genetic code! When an amino acid is linked to a tRNA, it will be incorporated into a growing protein.This step takes place in the cytosol Each aa is covalently attached to a specific tRNA, ATP is used.Enzyme is Mg-dependent aminoacyl-tRNA synthetase. R.S.UPENDRA RAJU Page 6 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY Components required for the activation of amino acids 1) 20 amino acids. 2 ) 20 aminoacyl-tRNA synthetases 3) 32 or more tRNAs.4 ) ATP. 5 ) Mg2+ . Aminoacyl-tRNA molecules made in 2 steps 1. Formation of aminoacyl adenylate 2. Attachment of aminoacyl group to the correct t-RNA molecule. Overall reaction: – Aa + tRNA + ATP--------> Aminoacyl-tRNA + AMP + PPi Translation Initiation Initiation mRNA require following components 1). N-Formylmethionyl-tRNAfmet, 2) Initiation codon in mRNA (AUG). 3) 30S & 50S ribosomal subunit 4).Initiation factors (IF-1, IF-2, IF-3). 5).GTP 6). Mg2+ R.S.UPENDRA RAJU Page 7 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY Signals for translation initiation Initiation sites in prokaryotic mRNAs are characterized by a Shine-Delgarno sequence that precedes the AUG initiation codon. Base pairing between the Shine-Delgarno sequence and a complementary sequence near the 3´ terminus of 16S rRNA aligns the mRNA on the ribosome. In contrast, eukaryotic mRNAs are bound to the 40S ribosomal subunit by their 5´ 7-methylguanosine caps. The ribosome then scans along the mRNA until it encounters an AUG initiation codon. Initiation of translation in bacteria Three initiation factors (IF-1, IF-2, and IF-3) first bind to the 30S ribosomal subunit. This step is followed by binding of the mRNA and the initiator N-formylmethionyl (fMet) tRNA, which is recognized by IF-2 bound to GTP. IF-3 is then released, and a 50S subunit binds to the complex, triggering the hydrolysis of bound GTP, followed by the release of IF-1 and IF-2 bound to GDP. R.S.UPENDRA RAJU Page 8 NEW HORIZON COLLEGE OF ENGINEERING DEPARTMRNT OF BIOTECHNOLOGY Elongation process and components essential for process 1).Functional 70S ribosome (initiation complex), 2) Aminoacyl-tRNAs specified by codons 3) Elongation factors (EF-Tu, EF-Ts, EF-G) . 4).GTP. 5) Mg2+ Elongation stage of translation The ribosome has three tRNA-binding sites, designated P (peptidyl), A (aminoacyl), and E (exit). The initiating N-formylmethionyl tRNA is positioned in the P site, leaving an empty A site. The second aminoacyl tRNA (AA 2) is then brought to the A site by EF-Tu (complexed with GTP).