Impact of pseudo-uridylation, substrate fold and degradosome organization on the endonuclease activity of RNase E
- 1 University of Cambridge Department of Biochemistry;
- 2 University of Wurzburg, Institute of Molecular Infection Biology;
- 3 University of Cambridge
- ↵* Corresponding author; email: bfl20{at}cam.ac.uk
Abstract
The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme’s hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multi-component RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5ʹ nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognised uracil is isomerised to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multi-enzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.
Keywords
- Received May 23, 2021.
- Accepted July 26, 2021.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
