Defining the mRNA recognition signature of a bacterial toxin protein

Abstract

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosomedependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide. (1,3,4). This rapid inhibitory switch suppresses metabolite consumption and temporarily halts cell growth to promote bacterial survival until nutrients are readily available. Among the prosurvival genes regulated by (p)ppGpp production are toxin-antitoxin modules, which have additional roles in antibiotic resistance and tolerance, biofilm and persister cell formation, and niche-specific colonization (5-11). The critical roles toxin-antitoxin pairs play in bacterial physiology underscore the importance of understanding their molecular targets and modes of action.There are five different classes (I to V) of toxin-antitoxin systems defined by how the antitoxin represses toxin function (1). Type II toxin-antitoxin pairs form protein-protein complexes during exponential growth that serve two purposes: inhibition of toxin activity by antitoxin binding and transcriptional autorepression to limit toxin expression (12). Antitoxins are proteolytically degraded after (p)ppGpp accumulation, leading to derepression at the toxin-antitoxin promoter (8, 12). Liberated toxin proteins inhibit the replication or translation machinery by targeting DNA gyrase, initiator tRNA fMet , glutamyl-tRNA synthetase, EF-Tu, free mRNA, ribosome-bound mRNA, and the ribosome itself (13-20).Ribosome-dependent toxins cleave mRNAs on the ribosome between the second and third nucleotides of the aminoacyl (A)-site codon (21-23). Although collectively Escherichia coli ribosome-dependent toxins target a diverse range of codons, each individual toxin appears to have a strong codon preference and cleaves at defined positions along the mRNA (24-26). RelE cleaves at UAG stop codons and the CAG sense codon (all codons denoted in the 5′-3′ direction); YoeB cleaves at codons following a translational AUG start site and at the UAA stop codon; and YafQ cleaves a single AAA sense codon (16,24,(27)(28)(29). In contrast, Proteus vulgaris host inhibition of growth B (HigB) toxin degrades multiple codons encod...

mRNA bound to the 30S subunit is a HigB toxin substrate

“…S1), but no interpretable electron density was observed for the mRNA in all four HigBmRNA soaks: wild-type HigB-wild-type mRNA; wild-type HigB-noncleavable mRNA; a catalytic HigB variant-wildtype mRNA; a catalytic HigB variant-noncleavable mRNA. The absence of mRNA in our structure is in contrast to all previous structures of the RelE, YoeB, and HigB toxins bound to the 70S ribosome (Neubauer et al 2009;Feng et al 2013;Schureck et al 2015), in which the mRNA is pulled ∼9 Å from its normal path in the A site into the active site of the toxin (Fig. 3B).…”

“…We attempted to soak into preformed 30S crystals combinations of wild-type HigB, a catalytic HigB variant (Hurley and Woychik 2009), wild-type mRNA (5 ′ -AAAUAG-3 ′ ), and a noncleavable mRNA (5 ′ -AAAUAG-3 ′ ) to program an AAA lysine codon in the A site and a UAG codon residing 3 ′ of the A site. In this mRNA fragment, the AAA lysine codon contains a 2 ′ -methoxy modification that our laboratory showed prevents cleavage (Schureck et al 2015). These approaches are similar to approaches previously used to observe 30S-bound ligands including IF1, 16S rRNA methyltransferase NpmA, and anticodon stem-loops of tRNAs bound to cognate and near-cognate codons of mRNAs Ogle et al 2001Ogle et al , 2002Dunkle et al 2014).…”

“…Given that no other 30S-toxin structures exist, this seems possible. Thus, although this crystal structure does not provide any direct insights into how HigB interacts with the mRNA when bound to a 30S complex, it demonstrates that HigB can bind the ribosome in the absence of the mRNA, informs on key A-site interactions with HigB, and lastly, allows us to compare the 30S-HigB orientation with that of a 70S-HigB elongation complex (Schureck et al 2015).…”

“…These observations suggest that the HigB endonuclease may target ribosomes during both initiation and elongation. We previously demonstrated that HigB recognizes a 70S elongation complex with the ribosome being essential for HigB-mediated mRNA cleavage (Schureck et al 2015).…”

“…We next compared the 30S-HigB structure with that of apo 30S and 70S-HigB structures caught in precleavage and postcleavage states (Wimberly et al 2000;Schureck et al 2015) (PDB codes 1J5E, 4YPB, and 4W4G). Comparison of the apo 30S and 30S-HigB structures reveals little to no changes indicating HigB does not induce large conformational rearrangement of the small subunit.…”