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 .
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• 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.
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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
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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
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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
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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.
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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+
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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.
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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). Following GTP hydrolysis, EF-Tu (complexed with GDP) leaves the ribosome, with (AA 2) tRNA inserted into the A site.
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Elongation I-
Elongation II
peptide bond is then formed, resulting in the transfer of methionine to the (AA 2 )tRNA at the A site. The ribosome then moves three nucleotides along the mRNA. This movement translocates the peptidyl (Met-AA 2) tRNA to the P site the uncharged tRNA to the E site, leaving an empty A site ready for saddition of the next aminoacid.
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Mechanism of Elongation II Mechanism of Elongation II
Elongation III
Translocation step The ribosome moves one codon toward the 3’ end of mRNA, using energy provided by GTP bound to translocase, EF-G. The movement of the ribosome shifts the dipeptidyl-tRNA from A site to the P site, and deacylated-tRNA is pushed back into the cytosol. This shift of the ribosome along the mRNA requires EF-G. This goes on and on. The ribosome moves from codon to codon along the mRNA toward 3’ end, adding one Amino acid residue at a time UNTIL…….
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Termination Elongation continues until the ribosome adds the last amino acid, completing the polypeptide chain coded by the mRNA. Termination signaled by one of STOP CODONS. UAA, AUG ,UGA There are 3 releasing factors: RF1, RF2, RF3. In eukaryotes, eRF recocnizes
Difference between Pro vs Eukaryotic protein synthesis
n Ribosomal units n Initiator n Start signal n Initiation – eIF vs IF n Elongation – EF1a, EF1bg n and termination factors – eRF
POST TRANSLATIONAL MODIFICATIONS (Folding and processing)
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Many proteins further processed by post-translational modification reactions both in prokaryotes and eukaryotes. Some of the important changes takes place @ PTM Amino and carboxy terminal modification • N-formyl Met, Met are removed. • Signal sequence removed. Modification of single aa. Attachment of carbohydrate side chains. Addition of prosthetic groups • Biotin is added to cty-c. Proteolytic process • Proteins are cleaved into small peptides, if necessary. Formation of disulfide links.
Effects of Post-translational Modification In generating the heterogeneity in proteins Help in utilizing identical proteins for different cellular functions in different cell types. Stability of protein. Biochemical activity (Activity regulation) Protein targeting (Protein localization) Protein signaling Most of the proteins that are translated from mRNA undergo chemical modifications before becoming functional in different body cells. The modifications collectively, are known as post-translational modifications. The protein post translational modifications play a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types.
Protein Post translational modifications May happen in several ways. Some of them are listed below: Glycosylation: Many proteins, particularly in eukaryotic cells, are modified by the addition of carbohydrates, a process called glycosylation.
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Glycosylation in proteins results in addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine. Acetylation: the addition of an acetyl group, usually at the N-terminus of the protein. Alkylation: The addition of an alkyl group (e.g. methyl, ethyl). Methylation: The addition of a methyl group, usually at lysine or arginine residues. (This is a type of alkylation.) Biotinylation: Acylation of conserved lysine residues with a biotin appendage. Glutamylation: Covalent linkage of glutamic acid residues to tubulin and some other proteins. Glycylation: Covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail of the amino acid sequence. Isoprenylation: The addition of an isoprenoid group (e.g. farnesol and geranylgeraniol). Lipoylation: The attachment of a lipoate functionality. Phosphopantetheinylation, The addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis. Phosphorylation, the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine. Sulfation: The addition of a sulfate group to a tyrosine. Selenation and C-terminal amidation .
Protein folding • Action of chaperones during translation Chaperones bind to the amino (N) terminal portion of the nascent polypeptide chain, • stabilizing it in an unfolded configuration until synthesis of the polypeptide is completed. • The completed protein is then released from the ribosome and is able to fold into its correct three-dimensional conformation.
Enzymes that Catalyze Protein Folding---The action of protein disulfide isomerase
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• Protein disulfide isomerase (PDI) catalyzes the breakage and rejoining of disulfide bonds, resulting in exchanges between paired disulfides in a polypeptide chain. • The enzyme forms a disulfide bond with a cysteine residue of the polypeptide and then exchanges its paired disulfide with another cysteine residue. • In this example, PDI catalyzes the conversion of two incorrect disulfide bonds (1-2 and 3-4) to the correct pairing (1-3 and 2-4).
Protein splicing -Proteolytic processing of insulin
• The mature insulin molecule consists of two polypeptide chains (A and B) joined by disulfide bonds. • It is synthesized as a precursor polypeptide (preproinsulin) containing an amino-terminal signal sequence that is cleaved during transfer of the growing polypeptide chain to the endoplasmic reticulum. • This cleavage yields a second precursor (proinsulin), which is converted to insulin by further proteolysis, removing the internal connecting polypeptide.
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Protein targeting: Signal hypothesis Steps in protein targeting I. Transmembrane Synthesis consists of following steps -Signal sequence and Signal Recognition Particle -Chaperone assisted protein folding -Glycosylation and protein targeting II. Nuclear Proteins targeting. Transmembrane Synthesis Translation of secreted proteins on free ribosomes results in a larger protein due to the presence of a signal sequence. The signal sequence directs the protein to the ER.Some ribosomes synthesize proteins directly into the ER. How does the ribosome know the protein should go to the ER? No surprise the signal sequence is involved. The signal sequence is bound by the Signal recognition particle (SRP) . SRP is a ribonucleoprotein which is a complex of 6 proteins and one RNA. SRP binding halts translation of the mRNA and directs the ribosome to the RER.
Transmembrane Synthesis Summary
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Signal recognition particle structure & function • Composed of one RNA and six proteins • It Binds to the signal sequence as soon as it emerges from the ribosome and Arrests translation Nascent Proteins in the ER fold back to its 3D conformation with the help of chaperons and Protein disulfide isomerase -Protein prolyl isomerase.
Signal hypothesis and cotranslational processing Coupling of transcription and translation in bacteria. The mRNA is translated by ribosomes while it is still being transcribed from DNA by RNA polymerase. This is possible because the mRNA in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes. In this schematic diagram the ribosomes are depicted as smaller than the RNA polymerase. In 6 reality the ribosomes (Mr 2.7 x 10 ) are an order of magnitude larger than the RNA polymerase (Mr 3.9 x 105). Glycosylation and Transport of protein Protein Glycosylation It is a Covalent addition of sugar residues to the protein. Carbohydrate addition - provide functional attributes -improves stability -provides transport signals to proteins.
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Proteins in ER must be transported to various compartments. Glycosylation plays a role in protein Targeting. Examples- Targeting of Membrane Proteins. - Secretory proteins-Plasma membrane proteins -Lysosomal proteins
Nucleus • Small proteins (<40 kDa) need no signal,Larger proteins require a signal • Nuclear targeting signal • Signal can appear anywhere in the protein, not just N-terminus
• Add a nuclear targeting signal to pyruvate kinase and it goes to the nucleus • Targeting signal is necessary and sufficient Protein Targeting Summary Peptides Sequences Target Proteins I. Signal Sequences for proteins targeted to ER, secretory, endosomal and plasma membrane proteins. - Glycoslyation serves to target proteins in secretory pathway. II. Signal Sequences for mitochondrial proteins, III. Nuclear transport requires a nuclear localization signal
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Comparing protein synthesis in prokaryotes and eukaryotes: a review Although bacteria and eukaryotes carry out transcription and translation in very similar ways, they do have differences in cellular machinery and in details of the processes. – Eukaryotic RNA polymerases differ from those of prokaryotes and require transcription factors. – They differ in how transcription is terminated, and how translation is initiated – Their ribosomes are also different. – One major difference: prokaryotes can transcribe and translate the same gene simultaneously. – The new protein quickly diffuses to its operating site.
In eukaryotes, the nuclear envelope segregates transcription from translation. In addition, extensive RNA processing is inserted between these processes. – This provides additional steps whose regulation helps coordinate the elaborate activities of a eukaryotic cell. In addition, eukaryotic cells have complicated mechanisms for targeting proteins to the appropriate point of need.
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TRANSLATION INHIBITORS
Protein synthesis can be inhibited by many antibiotics and toxins. Puromycin ---Premature chain elongation step. It is an analog of the terminal aminoacyl portion of aminoacyl-tRNA. It binds the A site but not to the P site. Tetracyclin ,Chloramphenicol ,Cycloheximide ,Streptomycin Dipheria toxin---It inhibits translocation. This toxin catalyzes the transfer of an ADP-ribose unit from NAD+ to diphthalomide, a modified amino acid residue in elongation factor 2 (translocase). Ricin.
Puromycin mechanism of action
Premature chain elongation step. It is an analog of the terminal aminoacyl portion of aminoacyl-tRNA. It binds the A site but not to the P site
Tetracycline n Mechanism of action. – Tetracycline antibiotics are protein synthesis inhibitors. – inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. – do so by binding to the 30S ribosomal subunit in the mRNA translation complex.
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Chloramphenicol
n Chloramphenicol is – a bacteriostatic by inhibiting protein synthesis. n Prevents protein chain elongation – by inhibiting the peptidyl transferase activity. n Specifically binds to – AA-2451 and AA-2452 residues in the 23S rRNA of the 50S ribosomal subunit, – preventing peptide bond formation
Cycloheximide
n Cycloheximide is a protein synthesis inhibitor in eukaryotes. n Its precise mechanism of action has yet to be fully elucidated. n inhibit translation elongation through binding to the E-site of the 60S ribosomal unit and interfering with deacetylated tRNA
Antibiotic Inhibitors of Translation
SINO ANTIBIOTIC INHIBITORY FUNCTION
1 Gentamicin, Erythromycin, Lincomycin Bind to 50S ribosomal subunit Chloramphenicol Inhibit peptide bond formation (translocation) 2 Neomycin, Kanamycin Bind to 70S subunit Prevent dissociation of 70S ribosomes; no translation 3 Streptomycin Bind to 30S subunit Inhibits initiation and causes misreading of mRNA 4 Tetracycline Bind to 30S subunit Prevents binding of aa-tRNA to mRNA ribosome complex 5 Puromycin Causes premature chain termination by acting as analog of aa-tRNA (both prokaryotes & eukaryotes) 6 Cycloheximide Inhibits translocation in eukaryotes
7 Ricin Irreversibly inactivates eukaryotic ribosomes by depurinating an A in 28S rRNA
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