Published online 15 July 2004
Nucleic Acids Research, Vol. 32 No. 12 © Oxford University Press 2004; all rights reserved
Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes
Yale University, Department of Molecular Biophysics and Biochemistry, 260 Whitney Avenue, New Haven, CT 06520 8114, USA, 1 Petersburg Nuclear Physics Institute, Gatchina 188355, Russia and 2 Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, 58448 Witten, Germany
* To whom correspondence should be addressed. Tel: +1 203 432 9772; Fax: +1 203 432 5767; Email: strobel{at}csb.yale.edu
Present address: A.E. Hesslein, Process and Technology Development, Bayer HealthCare, Berkeley, CA 94107, USA
Received April 14, 2004; Revised May 19, 2004; Accepted June 8, 2004
Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450•U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G•U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G•U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (kpep) was reduced 
200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G•U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pKa important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.
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