Fig The Elongation Cycle (in prokaryotes)

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) residues

PT loop Antibiotic footprints (circled bases) Antibiotic resistance mutations (circled bases) Antibiotics that inhibit PT bind to a loop in Domain V of 23S rRNA PT loop – peptidyl transferase loop

Locating the peptidyl transferase on the large ribosomal subunit

2 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 rd ed.

From Nissen et al., Science 289:920, S subunit from Haloarcula X-ray crystal structure Yarus analogue Fig RNA - grey proteins - gold

Nissen et al., Science 289: (2000)

From Nissen et al., Science 289:920, 2000 Also Fig in Weaver Active site: RNA + proteins Active site: only proteins, closest protein is at least 18 angstroms from the phosphate of the Yarus analogue. Fig

Evidence for rRNA as the PT 1.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

tRNA Charging: The Second Genetic Code 1.tRNA structure 2.the charging reaction 3.aminoacyl tRNA synthetases and tRNA recognition 4.proofreading mechanism

General 3D structure of tRNA Fig Variable loop

Fig

Amino acids are attached to the 3’ terminal nt of tRNAs (adenosine), via the 3’ or 2’ OH group. Amino acid portion 3’ term. A

tRNA 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 acid

Recognition 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, Biochemistry

Aminoacyl-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 loop

Fig GlnRS – tRNA Gln (Class I) AspRS-tRNA Asp (Class II) Class I - binds from the D loop side Class II – binds from the Variable loop side

How 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 methylene group (Isoleucine has 1 more) Accuracy achieved by the IleRS having 2 active sites: 1 st one activates most small amino acids (to aa-AMP) and the 2 nd one hydrolyzes the aa-AMPs smaller than Isoleucine (the editing site)

The double-sieve model for IleRS Fig