Adaptor protein RapZ activates endoribonuclease RNase E by protein–protein interaction to cleave a small regulatory RNA

Abstract

In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…GlmS makes glucosamine-6-phosphate (GlcN6P) required for the bacterial cell wall synthesis ( Milewski, 2002 ). When GlcN6P is abundant, a regulatory RBP, RapZ, captures GlmZ and delivers it to RNase E for cleavage; this prevents unnecessary GlmS production ( Göpel et al, 2013 , 2016 ; Durica-Mitic et al, 2020 ). But when GlcN6P is scarce, GlmY jumps in and titers RapZ out by chaining it in stable—and completely inert—class IIIb RNPs ( Gonzalez et al, 2017a ; Khan et al, 2020 ).…”

The nitrogen phosphotransferase regulator PtsN (EIIANtr) regulates inorganic polyphosphate production inEscherichia coli

“…The copyright holder for this preprint this version posted October 16, 2021. ; https://doi.org/10.1101/2021.10.15.464621 doi: bioRxiv preprint conditions where intracellular glucosamine-6-phosphate (GlcN6P) becomes limiting (29,31,54)(Fig. 3A).…”

“…1B). We therefore turned our attention to glmY, which, in a pathway involving the RNA-binding protein RapZ and the sRNA glmZ, is responsible for increasing GlmS (glutamine--fructose-6-phosphate aminotransferase) synthesis under conditions where intracellular glucosamine-6-phosphate (GlcN6P) becomes limiting (29,31,54)(Fig. 3A).…”

Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis

“…When GlcN6P levels are sufficiently high, GlmY is released by RapZ and degraded, allowing RapZ to bind GlmZ instead (Khan et al, 2020). Ultimately, RapZ presents GlmZ to the endoribonuclease RNase E in a manner that directs cleavage within the base pairing site of the sRNA, thereby inactivating it and silencing synthesis of the synthase (Göpel et al, 2013;Durica-Mitic et al, 2020;Gonzalez et al, 2017; Figure 1).…”

“…The quaternary structure of the RapZ tetramer is distinctive, with N and Cterminal domains forming separate symmetrical dimers lying at the apexes of the RapZ tetramer in a cyclic organisation (Figure 2b). Molecular genetics data indicate the requirement of the quaternary structure for stimulation of RNase E cleavage activity in vitro and overall regulatory activity in vivo (Gonzalez et al, 2017;Durica-Mitic et al, 2020). It is likely that the tetrameric organisation is required to present GlmZ for recognition and silencing by RNase E.…”