Lead‐ion‐induced cleavage of RNase P RNA

Abstract

Pbz+-induced hydrolysis of RNase P RNAs from Escherichia coli and the thermophilic eubacterium Thermus thermophilus HB8 revealed one prominent site-specific cleavage in the two RNAs and several minor cleavage sites in structurally corresponding regions of both RNAs. Data presented here and in a previous study [Kazakov, S. & Altman, S. (1991) Proc. Natl Acad. Sci. USA 88, 9193 -91971 provide evidence for several ubiquitous metal-ion-binding sites in eubacterial RNase P RNA subunits. With the I: thermophilus RNase P RNA, susceptibility to Pb2+-induced strand scission at the most prominent site was hypersensitive at the temperature of highest enzyme activity (55 "C). Pb" hydrolysis at this site was strongly reduced at a temperature of 37 "C, where processing is also inefficient. For E. coEi RNase P RNA, specific changes in the lead hydrolysis pattern were observed due to the presence of excess tRNA. Thus, Pb2+-induced hydrolysis seems suitable to sense different conformations of RNase P RNAs. The i ? thermophilus RNase P RNA, in particular, displayed significant processing activity after severe fragmentation by Pb2+, and therefore appears to be suited for reconstituting an active enzyme from RNA subfragments.The catalytic RNA subunits of eubacterial RNase P enzymes are sufficient in vitro to generate the mature 5' termini of precursor tRNAs (Guerrier-Takada et al., 1983). Cleavage of pre-tRNAs requires divalent cations, Mg" being most efficient (Guerrier-Takada et al., 1986. Though the essential role of divalent cations appears to be catalytic , they are likely to be further involved in maintaining spatial arrangements in RNase P RNA and RNA substrates (Holbrook et al., 1978), as well as in stabilizing enzyme-substrate complexes (Hardt et al., 1993). Metal-ion-induced cleavage of Escherichia coli RNase P RNA has been demonstrated to occur at five principal sites, and several metal ion cofactors such as Mn2+, Ca2+, S f ' and Ba2+, as well as metal ion inhibitors of RNase P RNA such as Zn", Coz+, Ni2+ and Cu2+, were shown to promote specific hydrolysis .The specific cleavage of yeast tRNAPh" between U17 and G18 by lead ions (Werner et al., 1976: Brown et al., 1983and 1985Rubin and Sundaralingam, 1983: Krzyzosiak et al., 1988; Behlen et al., 1990) has been exploited as a sensitive probe for the tertiary folding of tRNA variants (Behlen et al., 1990: Pan et al., 1991: Pan and Uhlenbeck, 1992 Dichtl et al., 1993). Beside highly specific Pbz+-induced hydrolysis due to the presence of tight metal-ion-binding sites, cleavages of lower intensity and specificity are observed at higher Pb2+ concentrations. It has been shown for several 5s rRNA molecules that cleavages occur mainly in singlestranded RNA regions, whereas helical regions are highly resistant to hydrolysis (Ciesiolka et al., 1992 a, b).Here we have analyzed lead-ion-induced hydrolysis of RNase P RNAs from E. coli and Thermus thermophilus HB8, an extreme thermophilic eubacterium (Oshima and Imahori, 1974). Preparation of RNAs E. coli RNase P RNA was transcri...

Mapping in Three Dimensions of Regions in a Catalytic RNA Protected from Attack by an Fe(II)-EDTA Reagent

“…When a substrate is employed (see below) that has only one very short stem-loop region, no protection of P4 and P8 is seen. We note also that while the Fe(II)-EDTA reagent did not detect large changes in the accessibility of C-4' atoms in the internal loop of P7 on binding of substrate, specific changes in susceptibility of this region to cleavage by divalent metal ions when substrate is bound have been observed (Kirsebom & Svard, 1993;Zito et al, 1993;Ciesiolka et al, 1994).…”

The precursor tRNA 3′-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo

“…In any case, the results of Figure 7 point to the sensitivity of RNase P RNA folding to structural changes as small as point mutations, particularly in low Mg 2+ buffers. This seems surprising in view of the highly dynamic nature of the loop L15 region, as inferred from biochemical experiments (Ciesiolka et al 1994;Brännvall et al 2003) and evident in the crystal structure of Thermotoga maritima RNase P RNA (Torres-Larios et al 2005). On the other hand, the dynamic nature of this region might increase its propensity to interfere with folding of other structural elements in the molecule.…”

“…Binding of tRNA CCA ends suppresses, in vitro and in vivo, prominent Pb 2+ hydrolysis at two locations in the L15 loop (sites III and V) and creates a new prominent Pb 2+ hydrolysis site (IVb) nearby (Ciesiolka et al 1994;Lindell et al 2005). This indicates a structural rearrangement of the P15/L15/P16 region upon formation of the G292-C 75 and G293-C 74 intermolecular base pairs.…”

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

“…In the current model, G291 forms a base triple with G259 in RNase P RNA and A76 in pre-tRNA; G292 forms a base triple with A258 in RNase P RNA and C75 in pretRNA; and U294 in RNase P RNA binds to R73 in pre-tRNA, where R is A or G (Kirsebom and Svard 1994;Heide et al 2001). There is evidence that J15/16 is also a metal-ion-binding site (Kazakov and Altman 1991;Zito et al 1993;Ciesiolka et al 1994;Kufel and Kirsebom 1994). Part of the mode of inhibition may be competing with the pre-tRNA for this binding site or interfering with metal ion binding.…”