Cs34: Specificity II - Specific aminoacylation of tRNA

1. NTP metabolism and transduction Cs34: Specificity II - Specific aminoacylation of tRNA Biochemistry 655 4 April 2011

2. Goals • Introduce the aminoacyl-tRNA synthetase class division • Polarity of substrate amino acids • Acceptor stem interactions - major/minor grooves and the Class I CCA3’ “hairpin”. • Dissect the anticodon binding site of GlnRS • Polar interactions • Nonpolar interactions • Specific base-pair recognition • Discuss what it means when the primary effects of anticodon mutations are to reduce kcat. • Relate the phenomenon of induced-fit to tRNA selection • Discuss the phenomenon of Kinetic proofreading of tRNA selection

3. Major Groove Minor Groove ~45 Å tRNA has approximate 2 symmetry

4. II Alanine Glycine Proline Serine Threonine Histidine Aspartic Acid Asparagine Lysine Phenylalanine I Leucine Valine Isoleucine Methionine Cysteine Arginine Glutamic Acid Glutamine Tyrosine Tryptophan Median DGxfr (H2O=>CHX) + 1.59 Kcal/mol - 2.97 { A } HIGH, KMSKS Motif 1, Motif 2 B C The aminoacyl-tRNA synthetase class distinction <D(DG)> ~3.6 kcal/mol => 400X • Eriani, et al., (1990) Nature, 347:203-206 • Cusack, et al., (1990) Nature, 347:249-255

5. Arg Glu Glu ATP aa aa Arg Gly Asp His ATP Gly Need Class I amino acids Lys Ser His Lys Ser Need Class II amino acids Class I and II active sites are unrelated… Class I active sites Class II active sites … but deeply interdependent

6. Class I and II bind tRNAs from opposite sides, and to opposite grooves GlnRS AspRS AspRS

7. Superposition of Gln, Asp complexes via the acceptor stem reveals extensive territorial respect!

8. Generalization by Ribas & Schimmel Ribas & Schimmel (2001) Cell, 104:191-193

9. Acceptor stem Anticodon KM has practically no role in specificity!

11. GlnRS squeezes the AC stem to expose it

15. Söll: in vivo evidence for discriminator base