The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.
Ribosomal variants carrying mutations in active site nucleotides are severely compromised in their ability to catalyze peptide bond formation (PT) with minimal aminoacyl tRNA substrates such as puromycin. However, catalysis of PT by these same ribosomes with intact aminoacyl tRNA substrates is uncompromised. These data suggest that these active site nucleotides play an important role in the positioning of minimal aminoacyl tRNA substrates but are not essential for catalysis per se when aminoacyl tRNAs are positioned by more remote interactions with the ribosome. Previously reported biochemical studies and atomic resolution X-ray structures identified a direct Watson-Crick interaction between C75 of the A-site substrate and G2553 of the 23S rRNA. Here we show that the addition of this single cytidine residue (the C75 equivalent) to puromycin is sufficient to suppress the deficiencies of active site ribosomal variants, thus restoring "tRNA-like" behavior to this minimal substrate. Studies of the binding parameters and the pH-dependence of catalysis with this minimal substrate indicate that the interaction between C75 and the ribosomal A loop is an essential feature for robust catalysis and further suggest that the observed effects of C75 on peptidyl transfer activity reflect previously reported conformational rearrangements in this active site.
Brunelle
Julie L
JL
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 702A PCTB, Baltimore, MD 21205, USA.
Youngman
Elaine M
EM
Sharma
Divya
D
Green
Rachel
R
D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010458:Peptidyl Transferases / Q000378:metabolism* / Q000493:pharmacokinetics*; D011691:Puromycin / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D012335:RNA, Ribosomal; D011691:Puromycin; D010458:Peptidyl Transferases