Lecture 3 Translation PDF

Lecture 3 Translation  polypeptide Transcription mRNA Tra export Translation DNA mRNA (in chromosome) Translati...

Lecture 3 Translation  polypeptide Transcription mRNA Tra export Translation DNA mRNA (in chromosome) Translation = mRNA is decoded by ribosomes to produce an amino acid chain (polypeptide) that will fold into an active protein Components needed for Translation Ribosome tRNA 60s 40s mRNA  Ribosome: factory site for protein synthesis Large Subunit = 5S, 5.8S, and 28S rRNA with 47 proteins (60S total) Contains the enzymatic activity (ribozyme) Small Subunit = 18S rRNA and 32 proteins (40S total) Responsible for reading the mRNA and monitoring complementarity between codon and anticodon  Ribosome: Aminoacyl-site: “charged” tRNA resides here Peptidyl-site: polypeptide chain resides here Exit-site: “uncharged” tRNA leaves ribosome here tRNA: carriers of amino acids Small RNAs (73-93 nbp); folded into 3-D structure Adapters in translating nucleic acids (mRNA) into proteins Structural features Amino acid arm (3’ end): Attaches to its specific AA; CCA residue at the end Each tRNA carries a specific AA 5’ end: Rich in poly guanylate (pG) residues Anticodon arm: Helps the tRNA to bind to the specific mRNA codon D arm: Has dihydrouridine (D) residues, an unusual base TΨC arm: Has unusual bases ribothymidine (T) & pseudouridine (Ψ) mRNA: contains genetic information 1. Composed of numerous codons arranged in linear, non-overlapping manner 2. Each codon codes for a specific AA 3. 64 (43) codons present 4. AUG (methionine): Initiation codon 5. UAA, UAG & UGA: Termination codons 6. 5’methylated cap & a 3’ poly A tail Note: Translation occurs in 5’ - 3’ direction How many bases required to code for an amino acid? Genetic code is degenerate 3’ 5’ G-A-A-G-A-G-G-A-T 5’ 3’ C-U-U-C-U-C-C-U-A Leucine Leucine Leucine Coding sequence is Linear & NON-overlapping 5’ 3’ A-U-U-C-U-C-C-U-A Isoleucine Leucine Leucine Coding sequence is Linear & NON-overlapping 5’ 3’ A-U-U-C-U-C-C-U-A … Phenylalanine Serine … Coding sequence is Linear & NON-overlapping 5’ 3’ A-U-U-C-U-C-C-U-A …. Serine Proline … Coding sequence is Linear & NON-overlapping 5’ 3’ A-U-U-C-U-C-C-U-A Isoleucine Leucine Leucine Genetic code is degenerate & almost universal The “Wobble” Hypothesis The “Wobble” Hypothesis The “Wobble” Hypothesis “Wobble” position 3’ 5’ tRNA G-G- I G-G- I G-G- I G-G-G 5’ 3’ mRNA C-C-U C-C-A C-C-C C-C-U  Translation Takes Place in 5 Stages Stage I - Activation of amino acids Stage II - Initiation Stage III - Elongation Stage IV – Termination & ribosome recycling Stage V – Folding & post translational modifications Note: Translation always occurs (read) in the 5’ - 3’ direction  Stage I – Activation of amino acids (charging tRNAs) Step 1: Amino acid “Activation” Step 2: tRNA conjugation Stage II – Initiation Steps involved 1.Dissociation of ribosome; binding of eukaryotic initiation factor 3 (eIF3) with the 40S subunit 2. Ternary complex: eIF2 & GTP binds to methionyl-tRNAMet 1 2 3 4 5 6 Stage II – Initiation 3. 43S pre-initiation complex: Ternary complex + 40S + eIF3+ eIF5+ eIF1 + eIF1a 4. Activation of mRNA: Facilitated by binding of cap binding complex called eIF4F (eIF4E+ eIF4G+ eIF4A) to the 5’ end of mRNA and then the 43S-pre-initiation complex binds to the eIF4F; scans mRNA for the initiation codon AUG Stage II – Initiation 5. Hydrolysis of GTP on eIF2 occurs (with help from eIF5); eIFs are 6 released 6. The 60S & 40S subunit bind placing AUG bound to methionyl-tRNAMet at the P site 7. The A site is open for the next codon coding the next AA 7  Regulation of the Initiation step Insulin stimulates protein synthesis by activating eIF4E of the cap binding complex Viral infections, starvation, heat shock, phosphorylates eIF2, inactivating it & inhibiting protein synthesis Stage III – Elongation Steps involved 1. Decoding The aminoacyl-tRNA (charged with next AA) binds to an elongation factor, eEF1A & GTP. The complex binds to the A site; hydrolysis of GTP occurs eEF1A-GDP complex dissociates from the ribosome eEF1A-GDP complex gets recharged with GTP in the cytosol Stage III – Elongation 2. Peptide bond Formation Occurs between carboxylic acid group of the AA in the P site and the amide group of the AA in the A site Catalyzed by peptidyl transferase (a rRNA with enzymatic activity) present in the 60S subunit Stage III – Elongation 3. Translocation eEF2 (bound to GTP) binds to the mRNA-ribosome complex GTP inducing conformational changes eEF2 The mRNA & its attached tRNAs move with respect to the ribosome GDP The uncharged (empty) tRNA moves eEF2 from P to E-site tRNA with the growing peptide moves from A to P site Uncharged tRNA exits from E-site; next aminoacyl-tRNA occupies the A site Stage IV – Termination Elongation continues until a stop codon enters the A site (UAA, UAG, UGA) Eukaryotic release factors eRF1 & eRF3 complex bind to the A site; eRF3 has GTPase activity Peptidyltransferase enzyme hydrolyzes the bond between the peptide chain & the tRNA at the P site releasing it; requires 1 GTP Ribosome dissociates into its subunits Translation proceeds 5’-3’ 80s ribosome 5’ Translation 3’ 5’-CAP mRNA 5’-end = amino terminus (N) 3’-end = carboxyl terminus (C) Met Gly Leu Ala Polypeptide  A Polyribosome complex Polyribosomes/Polysomes: Multiple ribosomes reading a particular mRNA simultaneously, synthesizing multiple copies of the same protein. As one ribosome moves along a mRNA translating it, a 2nd ribosome can bind to the 5” end of the mRNA  Stage V: Folding & Post Translational Modifications Folding & production of the 3-dimensional structure of protein Chaperons /heat shock proteins: Fold & refold the nascent (immature) polypeptide into its 3-dimensional conformation Disulfide isomerases: Introduce disulfide bonds stabilizing the folded structure Post translational modification: Imparts functionality by adding functional groups (methylation, glycosylation, hydroxylation, iodination, phosphorylation etc) Stage V – Folding and post translational modification Chaperons /heat shock proteins Disulfide isomerases and Post-translational Processing  Clinical Correlates Ribosomopathies: Congenital human disorders resulting from defects in ribosomal proteins/rRNA genes/other genes involved in ribosome biogenesis Ribosomes are prominent targets of many anti-bacterial drugs Diseases related to misfolded proteins Misfolded proteins generally get degraded immediately In some cases misfolded protein forms insoluble aggregates called amyloids Amyloids accumulate in the cells of different organs or extracellular space; associated with pathology of amyloidosis Alzheimer’s disease is an example of localized amyloidosis; caused by deposits of beta amyloid protein deposits (amyloid plaques) in the brain Creutzfeldt-Jakob Disease (CJD) Rare, degenerative, invariably fatal brain disorder A form of spongiform encephalopathy; 300 cases/year in the US Appears in 60+ years; aggressive, death within a year; may be sporadic/hereditary/acquired Prion proteins (causative agent of CJD) a. Glycoproteins b. Harmless when structurally normal (PrPc) c. Infectious when mis-folded (PrPsc) d. PrPsc is insoluble, resistant to protease digestion e. Cause of misfolding can be sporadic, genetic (dominant mutant PrPc gene) or transmitted f. One PrPsc can misfold several PrPc in a chain reaction (infectious)