A mechanistic study of peptide bond formation and peptide release on the ribosome
Catalysis of the related reactions of peptide bond formation and peptide release occurs in the highly conserved, all-RNA active site of the large ribosomal subunit. We have addressed the role of nucleotides present in the large subunit active site in catalysis using mutagenesis coupled with an affinity purification system that allows isolation of in vivo-assembled ribosomes bearing mutations in the ribosomal RNAs. Surprisingly, while mutation of four universally conserved active site nucleotides at structurally interesting positions caused substantial defects in the rate of peptide bond formation using a minimal substrate, no such defects were observed when intact substrates were used. These results strongly support a model where the ribosome catalyzes peptidyl transfer exclusively by aligning the reaction substrates in a pre-organized environment, an idea substantiated by subsequent results from a number of groups. While active site nucleotides are not important for peptidyl transfer, they do play a role in catalysis of peptide release, as mutations at these four nucleotides all led to reduced rates of peptide release.
Peptide bond formation and peptide release are catalyzed in the same active site and act on the same donor substrate, a peptidyl-tRNA, yet the hydrolytic peptide release reaction is promoted by protein release factors exclusively at stop codons. The mechanism by which this accuracy is achieved is poorly understood. We have demonstrated that, like the related process of tRNA selection, where the incorporation of cognate aminoacyl-tRNAs is promoted by specific structural rearrangements in the decoding center, stop codon recognition by release factors leads to conformational changes in the small subunit that we propose are productive for subsequent peptide release on the large subunit. These conformational changes are distinct from those promoted by tRNA selection during elongation.
This thesis demonstrates different roles for active site nucleotides in catalysis of peptide bond formation and peptide release, and provides insight into how the same active site can be regulated through distant interactions to perform two different catalytic functions.