"Off with Their (tRNA) Heads"

by Janie
 

Figure 1. (A) Cloverleaf view of a repre­sen­ta­tive tRNA. The sites of ribo­toxin cleav­age are marked with mag­enta arrows. More than one ribo­toxin may cleave tRNAs at the same site, but the iden­tities of their tRNA tar­gets could be dif­fer­ent. The ar­chaeal and euk­ar­yotic tRNA splic­ing site, as well as the tRNA cleav­age site by the euk­ary­otic ribo­some rescue fac­tors, is also mark­ed with blue arrows for com­pari­son. ColE5, coli­cin E5; ColD, coli­cin D; RQC, ribosome-assisted qual­ity con­trol. Source. (Note: there is a typo in the fig­ure: it should be Kp342, not Kp302.)

Some bacterial mole­cules have the social graces of certain 1700s French revol­ution­aries, or Shakespeare's Richard III, or the Queen of Hearts from Alice’s Adven­tures in Wonder­land – in that all share a pen­chant for behead­ing those they en­coun­ter! In the mic­rob­ial case, it's be­head­ing of tRNAs.
 

Among the arsenal of toxins dep­loyed in microb­ial turf squab­bles are endo­ribo­nucle­ases that chop off the 3' ends of tRNAs. The resul­ting "head­less" tRNAs can no longer carry amino acids and are no good for synth­esiz­ing proteins, to the great mis­for­tune of the cell. These butcher-toxins are part of the CdiA family of pro­teins, a collec­tion of des­truc­tive pro­teins used in con­tact-dependent growth inhib­ition. They come as a one-two punch: the aggres­sor cell secretes the CdiB/CdiA duo. The CdiB beta-barrel wedges into the victim's outer mem­brane to act as a back­door to its cyto­plasm. The CdiA effec­tor part­ner cleaves its own C-terminus – the part of the pro­tein that has teeth – and this CdiA-CT then slips through CdiB into the cell to do its cell-sabo­taging thing, whether that is des­troy­ing the cell's DNA, slic­ing up rRNA, or very com­mon­ly, going after tRNAs with guillotine-esque pur­poses.
 

CdiA-CTs are selec­tive going about their busi­ness. This is both in terms of which tRNA(s) are tar­geted, and what area on the tRNA is attack­ed, whether the anti­codon or T-loop or – as high­lighted here – the accep­tor stem. Take a CdiA-CT toxin from entero­hemor­rhagic E. coli, for ex­ample, called EC869, which prefers Gln and Asn tRNAs and cleaves them bet­ween the 71^st and 72^nd nucleo­tides. CdiA-CT^EC869 first com­man­deers the cell's own belong­ings: the two essen­tial trans­lation fac­tors EF-Tu and EF-Ts, plus GTP. The cata­lytic effi­ciency of CdiA-CT^EC869 soars when equip­ped with all three part­ners, form­ing a com­plex that then grabs onto its target tRNA. This broad over­view has been known since 2017 from a study by Jones et al.
 

Figure 2. Model of tRNA cleav­age by CdiA–CT^EC869 in the pres­ence of trans­la­tion fac­tors. The Tu:GTP:Ts com­plex acts as a scaf­fold for tRNA cleav­age by CdiA–CT^EC869. First, CdiA–CT^EC869 deliv­ered into the cell is rec­ruited to the Tu:GTP:Ts com­plex to form the CdiA–CT:Tu:GTP:Ts com­plex. Sub­strate aa-tRNA (or tRNA) is recog­nized by CdiA–CT:­Tu:GTP:­Ts and forms CdiA–CT:­Tu:­GTP:­Ts:­aa-tRNA(tRNA). Ts in the CdiA–CT:­Tu:­GTP:­Ts com­plex inc­reases the af­fin­ity of tRNA for the com­plex and in­duces a struc­tural change in tRNA and/or CdiA–CT to pro­mote prod­uc­tive cata­lysis by CdiA–CT. Asso­cia­tion of Ts to CdiA– CT:Tu:GTP or asso­cia­tion of CdiA–CT to Tu:GTP:Ts is req­uired for aa-tRNA(tRNA) bind­ing and cleav­age by CdiA–CT. Source.

But how does the toxin sniff out its tar­gets in the first place? A rec­ent study by Wang et al. did some more fine-toothed bio­chem­ical prob­ing into the pro­cess. First off, they noted that the tRNAs for Gln and Asn share a weak U1-A72 base pair at the top of the accep­tor helix. But so do the tRNAs for Trp (here, a weak A1-U72 pair) and f-Met (here, a C1-A72 mis­match). More­over, the Gln, Asn, and Trp tRNAs have a G73 as the dis­crimin­ator base (the base that for many tRNAs is im­por­tant in get­ting the cor­rect amino acid at­tached). As one might sus­pect, CdiA-CT^EC869 also chops up the Trp and f-Met tRNAs, sugges­ting that this flimsy tie at the ends of the tRNAs could be what dooms them. (Mean­while, chopping-resistant tRNA^Ala has a G-C base pair there instead – that's more of a sail­or's knot.) The toxin's recog­ni­tion of its speci­fic targets doesn't depend on the pres­ence of an amino acid; in terms of slic­ing and dic­ing effi­ciency, it doesn't care whether its tRNAs are amino­acylated or not. What is impor­tant here is the pres­ence of EF-Ts, which is req­uired for the toxin com­plex to bind a tRNA (a pull-down exper­iment with the toxin not bound to EF-Ts yields no tRNAs!).
 

A note: there is a dis­crep­ancy between the 2017 and 2022 studies. Jones et al. 2017 found that EF-Tu is absol­utely essen­tial for CdiA-CT^EC869 and that the toxin alone has no RNase activ­ity, where­as Wang et al. 2022 found that toxin activ­ity is still pres­ent at lower levels without EF-Tu, or EF-Ts, or GTP… Per­haps this just fur­ther high­lights the pH-dependence of the toxin: the 2022 study tested for RNase activ­ity in a pH range from 5.3 to 8.1, and found that depen­dence on EF-Tu/EF-Ts/GTP inc­reased as pH inc­reased. At low pH, there was barely any dif­fer­ence in activ­ity bet­ween the toxin alone and the toxin in-complex, while at a higher pH, the toxin alone had almost no activ­ity. Fit­tingly, the 2017 study did their assays ar­ound neut­ral pH. And, to ac­count for the bit of 2022 solo-toxin activ­ity at sim­ilar higher pH, perhaps there was a bit of contam­inat­ing EF-Tu or EF-Ts stuck to the puri­fied toxin or tRNAs.
 

Finally, there is a region of CdiA-CT^EC869 lined with sev­eral posi­tively charged resi­dues. This spot makes for a nice rest­ing place (in both senses of the noun…) for the nega­tively charged RNAs. It is here, the authors pro­pose, that the 3' end of the tRNA is held, and then chop­ped. Vive la révol­ution…