title: Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes.
abstract: 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 (k(pep)) was reduced approximately 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 pK(a) important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.
authors.affiliation: Yale University, Department of Molecular Biophysics and Biochemistry, 260 Whitney Avenue, New Haven, CT 06520 8114, USA.
authors.forename: Ashley Eversole
authors.initials: AE
authors.lastname: Hesslein
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Katunin
authors.forename: Malte
authors.initials: M
authors.lastname: Beringer
authors.forename: Anne B
authors.initials: AB
authors.lastname: Kosek
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D000225:Adenine / Q000737:chemistry*; D020029:Base Pairing; D001483:Base Sequence; D002846:Chromatography, Affinity; D003596:Cytosine / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010458:Peptidyl Transferases / Q000378:metabolism*; D017354:Point Mutation; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology / Q000235:genetics*; D014498:Uracil / Q000737:chemistry
chemical_list: D012338:RNA, Ribosomal, 23S; D014498:Uracil; D006147:Guanine; D003596:Cytosine; D010458:Peptidyl Transferases; D000225:Adenine