Residues inEscherichia coliRNase P RNA Important for Cleavage Site Selection and Divalent Metal Ion Binding

The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain

“…We also investigated whether the GGU-motif in the lower half of the P15-loop, which in M1 RNA base pairs with the 39-terminal RCCA sequence of a pre-tRNA (Kirsebom & Svärd, 1994;Svärd et al+, 1996;Tallsjö et al+, 1996), is accessible for base pairing in the case of the 31-mer RNA+ To assess this, an excess (5-50-fold) of a short DNA oligonucleotide, 59-CACCCA, carrying four nucleotides complementary to G23-U26 (corresponding to G291-U294 in M1 RNA) was hybridized under native conditions to the 31-mer RNA+ Subsequent addition of RNase H resulted in cleavage 39 of U26 (Fig+ 3) showing that a DNA-RNA hybrid had been formed+ In an analogous experiment using the same DNA oligonucleotide and a mutant version of the 31-mer RNA carrying a G to C change at position 25, no RNase H cleavage was observed+ However, when another DNA oligonucleotide (59-CAGCCA) complementary to the mutant derivative was used, cleavage by RNase H using the C25 version was detected+ These findings suggest that G24, G25 (or C25), and U26 are accessible for base pairing, in keeping with similar results obtained using full-length M1 RNA (Kufel & Kirsebom, 1996)+ The accessibility of these three residues is expected from the NMR-based structure of the 31-mer RNA where they are exposed on the surface of the molecule (Glemarec et al+, 1996; Fig+ 6A)+ Upon binding of the DNA oligonucleotide, we also observed that the N7 position of A5 became even less accessible (if at all) to modification with DEPC (data not shown) compared to the modification pattern in the absence of this DNA oligonucleotide+ This observation may indicate that formation of the DNA-RNA hybrid is accompanied with a conformational change(s) of the neighboring resi-dues in the loop+ This would be consistent with our previous hypothesis that the "RCCA-M1 RNA" interaction induces conformational changes within the enzyme-substrate complex, in particular in the P15-loop (Kufel & Kirsebom, 1996)+ In this context, we also note that a structural model of the P15-loop in complex with the 39-terminal RCCA-motif of the substrate was presented recently (Easterwood & Harvey, 1997)+ This model shows similarities with the structure of the P15-loop as determined by NMR (Glemarec et al+, 1996; see below), however, there are also obvious differences+ Some of these might indeed be due to conformational changes induced by enzyme-substrate complex formation+ Together, these data further strengthen the conclusion that the P15-loop structure is similar within both the 31-mer RNA and M1 RNA+ This makes the 31-mer RNA a useful model molecule representing the P15-loop of M1 RNA+…”

“…A: Secondary structure model of E. coli RNase P RNA (M1 RNA) according to Brown (1996)+ Specific Pb 2ϩ -induced cleavage sites are indicated by arrows and roman numerals, whereas Mg 2ϩ -induced cleavage sites are shown by numerals in italics+ Shaded nucleotides represent residues that base pair with the 39-terminal RCCA-motif of a precursor (Kirsebom & Svärd, 1994)+ B: Schematic illustration of the 31-mer RNA carrying the P15-loop of M1 RNA+ Arrows indicate Mg 2ϩ -and Pb 2ϩ -induced cleavage, with dashed arrows for unique Pb 2ϩ -induced cleavage sites in the tertaloop+ A line dividing the molecule into two halves indicates the site where the two RNA fragments were ligated to generate "semispecifically" substituted molecules containing modifications in either of the halves (see Materials and Methods)+ Grey box indicates cleavage of RNA-DNA hybrid by RNase H+ Substitutions at positions 8, 9, and 25 were made as indicated (U8, A9, and G25 correspond to U257, A258, and G293, respectively, in full-size M1 RNA)+ Shaded nucleotides depict residues in intact M1 RNA involved in base pairing with the substrate+ served 39 of G25 and U26 are in agreement with binding of metal ion(s) in this region as predicted by NMR studies (Glemarec et al+, 1996)+ In the presence of higher concentrations of Pb 2ϩ , additional cleavage sites in the tetraloop were observed (e+g+, see Fig+ 2A, lane 3)+ The absence of Mg 2ϩ -induced cleavage at these sites does not exclude the possibility of a Mg 2ϩ ion(s) being present in the vicinity of this region+ In fact, the NMR data suggest a Mg 2ϩ binding site in the vicinity of G18 (Glemarec et al+, 1996), but this (or these) Mg 2ϩ does not promote cleavage+ In any case, this metal ion binding site is not relevant for the function of M1 RNA, because the tetraloop is not present in M1 RNA at this position+ In conclusion, this short RNA molecule represents an autonomous divalent metal ion binding domain of M1 RNA+ This finding suggests that the P15-loop within the 31-mer forms a structure that is comparable to that of the equivalent loop in full-length M1 RNA+ The P15-loop within the 31-mer RNA is a model molecule for the corresponding region in M1 RNA Substitutions of a guanosine to adenosine and cytosine at position 293 in M1 RNA reduced Pb 2ϩ -induced cleavage at site V, whereas cleavage at site III was not affected (Kufel & Kirsebom, 1996; see Fig+ 1; and data not shown)+ If the structure of the P15-loop in the 31-mer RNA is the same as in the full-length M1 RNA, it is expected that substitutions of G25 (corresponding to position 293 in M1 RNA, see Fig+ 1B) would affect divalent metal ion cleavage 39 of this position+ As shown in Figure 2B, cleavage with Mg 2ϩ at this site (V) was clearly decreased when the mutated derivatives of the 31-mer RNA were used, whereas cleavage at site III was not altered+ Identical results were also observed in the case of cleavage with Pb 2ϩ (data not shown)+ Similar analogy between the 31-mer RNA and M1 RNA 6) and of various derivatives mutated at position 25 (A25, lanes 2 and 7; U25, lanes 3 and 8; C25, lanes 4 and 9) or at positions 8 and 9 (G8G9, lanes 5 and 10)+ MgCl 2 was added to a final concentration of 10 mM as indicated+ C: Magnesium(II)-induced cleavage of wild-type (wt) 31-mer RNA (lanes 2 and 5) and its derivatives substituted with deoxyU in the lower (dUl; lanes 3 and 6) and upper (dUt; lanes 1 and 4) half of the molecule+ MgCl 2 was added to a final concentration of 10 mM as indicated+ was observed in the case of changes in the upper part of the P15-loop+ The main Mg 2ϩ -induced cleav...…”

“…RNase H cleavage of RNA-DNA oligonucleotide hybrids+ Cleavage reactions were performed as described in Materials and Methods using 32 P 59 end-labeled wild-type (wt) 31-mer RNA (lanes 1-3) or its "C293" derivative (lanes 4-6)+ A short DNA oligonucleotide (59-CACCCA or 59-CAGCCA) was allowed to hybridize to the RNA molecules under native conditions at 33 8C followed by addition of RNase H+ Cleavage products and uncleaved RNAs were separated on a 20% denaturing polyacrylamide gel+ Arrow indicates the RNase H cleavage observed in the lower half of the internal loop 39 of U26 [corresponding to U294 in M1 RNA (Kufel & Kirsebom, 1996)]+ RNase H and DNA oligonucleotides were added as indicated+ L represents partial alkaline ladder of 59 end-labeled 31-mer RNA+ Lanes 1, 4, no DNA oligonucleotide was added; lanes 2, 5, a DNA oligonucleotide, complementary (C) to residues G23-U26 of either wild-type (59-CACCCA) or the "C293" derivative (59-CAGCCA) was added; lanes 3, 6, DNA oligonucleotide not fully complementary (NC) to the same region of either wild-type (59-CAGCCA) or "C293" derivative (59-CACCCA) was added+ cations within the P15-loop+ [Mg 2ϩ or Pb 2ϩ coordinated in this way would subsequently result in cleavage 59 of U26 and U8 (see Fig+ 1B)+] Metal ions coordinated to these nonbridging oxygens in each half of the molecule are important for metal ion-induced cleavage only in the respective half without affecting cleavage in the other half+ We note that the sequence 59-PuUA is present at both these sites and at another metal ion-induced cleavage site in full-length M1 RNA, site IV (59-AUA, located in a 4-nt bulge joining two helical stems, see Fig+ 1), as well as at metal ioninduced cleavage sites in Bacillus subtilis RNase P RNA (Zito et al+, 1993) and in a Group I intron (Streicher et al+, 1996)+ Taken together, this might suggest that the 59-PuUA sequence plays a role in divalent metal ion binding, however, we have to await further experiments using other RNA molecules in order to conclude whether this is the case or not+ Furthermore, magnesium(II)-induced cleavage 59 of, for example, A9 and A27 appears not to be affected by their Rp diastereomers (Fig+ 4A)+ In the proposed mechanism for the hammerhead ribozyme cleavage, direct coordination of Mg 2ϩ to pro-Rp oxygen at the cleavage site most likely takes place in the transition state of the reaction (Scott et al+, 1995(Scott et al+, , 1996+ Our data suggest that this may differ in the case of coordination of metal ions involved in cleavage at these positions in the 31-mer RNA+ Thus, another way of coordinating catalytic Mg 2ϩ in the vicinity of these particular sites, for example using pro-Sp oxygen as a ligand, could be responsible for cleavage at these sites+ Similar analysis of the RNA molecules substituted with thio-G was not conclusive because, although cleavage with Pb 2ϩ was stimulated 39 of A9 and G10, the reduction of the Mg 2ϩ -induced cleavage at these sites was less clear (Fig+ 4B)+ The cleavage pattern of the thio-C-substituted RNA did not exhibit any significant differences compared to the natural 31-mer RNA (data not shown)+ This is expected because no major divalent metal ion cleavage sites are present in the vicinity of the cytidines+…”

Metal ion cooperativity in ribozyme cleavage of RNA

“…Base substitutions in the P15 loop, a domain of M1 RNA that interacts with the RCCA motif at the 3Ј end of the substrate, the RCCA-RNase P RNA interaction (interacting residues underlined; Fig. 1b), result in reduced activity and changes in divalent metal ion binding in M1 RNA (16,22). Thus, we decided to combine the chemical approach discussed above with a genetic approach to study whether the divalent metal ion(s) bound in P15 (Fig.…”

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA

“…Since gel-resolvable tRNA-binding to RNase P RNA substantially relies on the NCCA interaction (Hardt et al+, 1995a), our interference assay seems to be highly sensitive to minor destabilizations in the P15/16 domain+ Our observation of 29-deoxy modifications at C253 and A256 that interfere with tRNA binding may also reflect a direct or indirect role of the 29-hydroxyls at these positions in metal ion coordination or in stabilizing tertiary contacts between nucleotides in the J15/16 internal loop and other regions of RNase P RNA+ In summary, it has become clear that the structural integrity of the J15/16 loop is tightly linked to its metal-ion binding properties and to the alignment of the cleavage site (Kufel & Kirsebom, 1996b;Westhof et al+, 1996)+ The NCCA interaction seems to induce a structural rearrangement of the RNase P RNA-(p)tRNA complex including a recoordination of metal ion(s) in the vicinity of the cleavage site (Ciesiolka et al+, 1994;Kufel & Kirsebom, 1996b;Westhof et al+, 1996;Oh et al+, 1998)+ Base, ribose, and phosphate moieties of nucleotides in the upper and lower parts of the J15/16 loop as well as base and ribose moieties of the tRNA NCCA terminus influence this process (Perreault & Altman, 1992;Kufel & Kirsebom, 1996bTallsjö et al+, 1996;Oh et al+, 1998)+ Combined Rp-phosphorothioate and inosine interference effects were observed at G250 and G300 (Table 1)+ The Rp-GMPaS interference at the phylogenetically conserved G300 is enhanced by additional inosine (Table 1) or 29-deoxy modifications + A G300-to-C mutation largely decreased tRNA binding affinity to E. coli RNase P RNA, although binding affinity could be partially restored at high monovalent salt concentrations+ The G300-to-C mutation also caused differences in the susceptibility to lead hydrolysis in the J5/15 to P17 region + In summary, G300 is critical for (p)tRNA binding and several functional groups of this nucleotide seem to be involved in an intricate, yet uncharacterized, network of interactions+ Considering the just-mentioned ); however, in these two cases, Ca 2ϩ was substituted for Mg 2ϩ and the pH was reduced from 7+0 to 6+0 to avoid processing of ptRNA during preincubation and electrophoresis (for details, see Materials and Methods)+ phenotype of the G300-to-C mutant, the universal conservation of G300 among bacterial and archaeal RNase P RNAs (RNase P database; Brown, 1998), and taking into account the two available models of the RNase P RNA-(p)tRNA complex (Chen et al+, 1998;Massire et al+, 1998), it is likely that G300 affects (p)tRNA binding indirectly by playing a crucial role in folding of the core structure, rather than forming a direct contact to the tRNA moiety+ Phylogenetic comparative analysis predicts that G250 forms a G-U pair with U299 in E. coli RNase P RNA+ G250 is part of the core structure and several crosslinks with (p)tRNAs carrying a photoreactive group near the...…”