Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit.
On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length beta-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro beta-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.
Thompson
J
J
Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Jill_Rosemary_Thompson@brown.edu
Kim
D F
DF
O'Connor
M
M
Lieberman
K R
KR
Bayfield
M A
MA
Gregory
S T
ST
Green
R
R
Noller
H F
HF
Dahlberg
A E
AE
D001665:Binding Sites; D002701:Chloramphenicol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016297:Mutagenesis, Site-Directed; D010458:Peptidyl Transferases / Q000378:metabolism*; D010641:Phenotype; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics*
D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D002701:Chloramphenicol; D010458:Peptidyl Transferases