PowerPoint PresentationFootprinting drug binding sites on rRNA (Moazed & Noller)Slide 3Locating the peptidyl transferase on the large ribosomal subunitSlide 5Slide 6Slide 7Slide 8Evidence for rRNA as the PTSlide 10Slide 11tRNA Charging: The Second Genetic CodeSlide 13Slide 14Slide 15tRNA ChargingRecognition of tRNAs by Aminoacyl-tRNA synthetases: the Second Genetic CodeAminoacyl-tRNA synthetases (cont.)Slide 19How is charging accuracy achieved, given the structure of amino acids?Slide 21Fig. 18.10The Elongation Cycle (inprokaryotes)Footprinting drug binding sites on rRNA (Moazed & Noller)•Analogous to footprinting a protein binding site on DNA or RNA•Can map where antibiotics bind to rRNA in the ribosome •Bound drug prevents chemical modification of the bases (use DMS for purines and CMCT for U)•Modified bases cause reverse transcriptase to stop during primer extension; doesn’t stop at unmodified (protected) residuesPT loopPT loopPT loopAntibiotic footprints(circled bases)Antibiotic resistance mutations(circled bases)Antibiotics that inhibit PT bind to a loop in Domain V of 23S rRNAPT loop – peptidyl transferase loopLocating the peptidyl transferase on the large ribosomal subunit2 analogues (b and c) that should bind to the active site of PT on the large ribosomal subunit (b) resembles the transition state formed during the real reaction (a)(c) resembles a substrate and docks into the A site“Yarus analogue”Fig. 19.21 3rd ed.From Nissen et al., Science 289:920, 200050S subunit from HaloarculaX-ray crystal structureYarus analogueFig. 19.16RNA - greyproteins - goldNissen et al., Science 289: 920-930 (2000)From Nissen et al., Science 289:920, 2000Also Fig. 19.25 in WeaverActive site: RNA + proteins Active site: only proteins, closest protein is at least 18 angstroms from the phosphate of the Yarus analogue.Fig. 19.17Evidence for rRNA as the PT1. No ribosomal proteins have been identified that have peptidyl transferase (PT) activity.2. Drugs (e.g., Chloramphenicol) that inhibit PT bind to the 23S rRNA, in the PT loop of Domain V.3. Mutations that provide resistance to the drugs that inhibit PT map to the same loop.4. Nearly all (99%) of the protein can be stripped from the 50S subunit, and still have PT activity. 5. The X-ray crystal structure of the 50S subunit shows that only RNA chains (PT loop, etc.) are close enough to catalyze a reaction.• Are there any potential deficiencies with this model or the data that support it?• How could it be made stronger?Fig. 19.28tRNA Charging: The Second Genetic Code1. tRNA structure2. the charging reaction3. aminoacyl tRNA synthetases and tRNA recognition4. proofreading mechanismGeneral 3D structure of tRNAFig. 19.26Variable loopFig. 19.25Amino acids are attached to the 3’ terminal nt of tRNAs (adenosine), via the 3’ or 2’ OH group.Amino acid portion3’ term. AtRNA Charging•Occurs in two steps:1. AA + ATP Aminoacyl-AMP + PP 2. Aminoacyl-AMP + tRNA Aminoacyl-tRNA + AMP•Catalyzed by Aminoacyl-tRNA synthetases•Cells must have at least 20 aminoacyl-tRNA synthetases, one for each amino acidRecognition of tRNAs by Aminoacyl-tRNA synthetases: the Second Genetic Code Aminoacyl-tRNA synthetases recognize mainly the acceptor stem and the anticodon.From Voet and Voet, BiochemistryAminoacyl-tRNA synthetases (cont.)•Diverse group of enzymes despite recognizing fairly similar substrates•Not well conserved, however there are 2 main classes–Class I (aminoacylate the 2’ OH) –Class II (aminoacylate the 3’ OH)•Each class has the same 10 members in all organisms•The classes bind tRNA somewhat differently, but both bind to the acceptor stem and the anticodon loopFig. 19.30GlnRS – tRNAGln (Class I) AspRS-tRNAAsp (Class II)Class I - binds from the D loop sideClass II – binds from the Variable loop sideHow is charging accuracy achieved, given the structure of amino acids?•Isoleucine tRNA synthetase (IleRS is Class I) discriminates > 50,000-fold for Ile over valine •Ile and Val differ by only one methylenegroup (Isoleucine has 1 more)•Accuracy achieved by the IleRS having 2 active sites: 1st one activates most small amino acids (to aa-AMP) and the 2nd one hydrolyzes the aa-AMPs smaller than Isoleucine (the editing site)The double-sieve model for IleRSFig.
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