Substrate-dependent effects of quaternary structure on RNase E activity

Abstract

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5′ terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Sodium Fluoride Exposure Leads to ATP Depletion and Altered RNA Decay in Escherichia coli under Anaerobic Conditions

“…4Ba and Bb and 4Fa and Fb ), it seems likely that this effect could be caused, at least in part, by inhibition of their RhlB-dependent and/or PAPI-dependent decay, presumably due to NaF-dependent ATP depletion. Moreover, degradation of both transcript types may be dependent on RNase E, the key player in E. coli mRNA decay, whose quaternary structure regulates RNA turnover in a substrate length-dependent manner ( 44 ). Indeed, our assessment of RNA half-lives in wild-type (N3433) and temperature-sensitive RNase E mutant (N3431) strains revealed enhanced stability of osmC and yghA transcripts upon inactivation of RNase E (i.e., in N3431 at the nonpermissive temperature).…”

A conserved protein inhibitor brings under check the activity of RNase E in cyanobacteria

“…Due to its broad substrate specificity, its activity must be under check according to changes of internal or external conditions. Yet, only a few regulators of RNase E have been described (Lee et al ., 2003; Singh et al, 2009; Górna et al, 2010; Moore et al, 2021), and most of them are present only in a limited number of bacteria, suggesting that distinct regulation mechanisms may have been evolved in different organisms. Here, we report that RebA, a protein universally present in cyanobacteria, could inhibit the activity of RNase E. RebA was co-isolated with RNase E from Anabaena cell lysate by co-immunoprecipitation.…”

“…The activity of RNase E is regulated in multiple ways, including subcellular localization, transcriptional autoregulation, and formation of RNA degradosome (Jain and Belasco, 1995; Prud’homme-Généreux et al, 2004; Murashko et al, 2012; Zhou et al, 2020). Additionally, RNase E activity can also be regulated by transacting components, such as the E. coli proteins of RraA, RraB, ribosomal L4 protein, AmiC (Lee et al, 2003; Gao et al, 2006; Singh et al, 2009; Moore et al, 2021). These regulators mostly associate with the noncatalytic region of RNase E, and either enhancing or disrupting their interactions with RNase E did not severely affect cell growth and RNA metabolism of the corresponding bacteria, suggesting their limited regulatory effects in vivo .…”

“…Dip, a protein from the giant phage фKZ, could inhibit the activity of RNase E of its host Pseudomonas aeruginosa by binding to the RNA binding sites in the noncatalytic region (Van den Bossche et al, 2016). Recently, a bacterial cell wall peptidoglycan hydrolase, AmiC, was shown to stimulate the activity of RNase E, potentially by enhancing RNase E multimerization (Moore et al, 2021). It is yet unknown if any of these regulation mechanisms exist in the other RNase E-containing organisms.…”

A conserved protein inhibitor brings under check the activity of RNase E in cyanobacteria

“…Dip, a protein from the giant phage фKZ, could inhibit the activity of RNase E of its host Pseudomonas aeruginosa by binding to the RNA binding sites in the noncatalytic region ( 35 ). Recently, a bacterial cell wall peptidoglycan hydrolase, AmiC, was shown to stimulate the activity of RNase E, potentially by enhancing RNase E multimerization ( 36 ). It is yet unknown if any of these regulation mechanisms exist in the other RNase E-containing organisms.…”