The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.
Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.
Youngman
Elaine M
EM
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
Brunelle
Julie L
JL
Kochaniak
Anna B
AB
Green
Rachel
R
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D011500:Protein Synthesis Inhibitors; D012346:RNA, Transfer, Amino Acyl; D011691:Puromycin; D008274:Magnesium