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Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide

Nature volume 411, pages 498–501 (2001)Cite this article

Abstract

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the α-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 Å from the active site, revealing peptidyl transferase as an RNA enzyme1. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue2,3. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.

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Figure 1: The secondary structure of the central loop of domain V of T. aquaticus 23S rRNA.
Figure 2: Peptidyl transferase activity of reconstituted large ribosomal subunits harbouring mutations of the proposed catalytic nucleotide A2451.
Figure 3: The time course of peptidyl transferase reactions catalysed by reconstituted 50S subunits containing mutations at position 2451.

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Acknowledgements

We thank M. Gomez and L. Xiong for help with experiments; S. Swaney and D. Shinabarger for providing SPA assay protocols and reagents; and J. Piccirilli, A. Mesecar and A. Neyfakh for helpful discussions. This work was supported by an NIH grant to A.S.M. N.P. was supported by an Erwin Schroedinger fellowship from the Austrian Science Foundation.

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  1. Center for Pharmaceutical Biotechnology (MC 870), University of Illinois, 900 South Ashland Avenue, Chicago, 60607, Illinois, USA

    Norbert Polacek, Marne Gaynor, Aymen Yassin & Alexander S. Mankin

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Correspondence to Alexander S. Mankin.

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Polacek, N., Gaynor, M., Yassin, A. et al. Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature 411, 498–501 (2001). https://doi.org/10.1038/35078113

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