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

Abstract

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH 2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH 2 groups of G292/G293 in pairing with C 74 and C 75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C 75 and A 76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA. Keywords: gel retardation; inosine interference; mutations in loop L18 and helix P8; nucleotide analog interference mapping (NAIM)

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

“…Runoff transcription with bacteriophage T7 RNA polymerase as well as 59 and 39 endlabling were performed essentially as described (Heide et al 1999;Busch et al 2000). The ptRNA Gly substrate (Fig.…”

Purine N7 groups that are crucial to the interaction of Escherichia coli RNase P RNA with tRNA

“…Sites of c7-deaza interference in E. coli RNase P RNA Sp-stereoisomers of c7-deaza-GTPaS (Fig+ 1) and c7-deaza-ATPaS used for T7 transcription were synthesized as described (Kazantsev & Pace, 1998)+ C7-deazapurine analogs lack the hydrogen bonding and metal-coordinating properties of purine N7 positions+ For enzymatic synthesis of partially modified pools of RNase P RNA, 50 mM of the RTPaS or c7-RTPaS analog (R ϭ A or G) and 1 mM of the corresponding unmodified nucleotide were included during T7 transcription to achieve a low level of modification (Kazantsev & Pace, 1998)+ As in our previous NAIM studies (Hardt et al+, 1995bHeide et al+, 1999), partially modified E. coli RNase P RNA pools (labeled at either the 59 or 39 terminus) were separated into tRNA-binding and nonbinding fractions by gel retardation and then subjected to iodine cleavage (Heide et al+, 1999)+ At sites of interference, the radioactivity of the corresponding iodine hydrolysis product was found to be reduced in the tRNA-binding RNase P RNA fraction and increased in the nonbinding fraction+ Interference patterns observed in the presence of the c7-RMPaS double modification were then compared with those of the RMPaS modification alone to assign interference effects to the phosphorothioate (termed thioate in the following text) and/or the c7-deaza modification+ An inherent limitation of this approach is that strong thioatespecific interference effects will prevent analysis of any additional modification+ Comparative iodine hydrolysis patterns of the two separated E. coli RNase P RNA fractions are illustrated in Figure 2 and summarized in Figure 3+ Only a few c7-deazaguanine interference effects were observed+ Sites of strong c7-specific interference (.50%) could be assigned to G292 and G306 (Fig+ 3)+ Thioate-specific interference at G250/251 (Heide et al+, 1999) was slightly enhanced in the presence of the additional c7-deaza modification (Fig+ 3)+ Band compression at positions 250/251 prevented the final assignment of interference to G250 or G251, although the predominant effect appeared to be located at G250 (Heide et al+, 1999)+ Strong c7-deazaadenine interference effects (.50%) were identified at A65, A136, A234, A305, A334, and A351+ Effects of intermediate strength (25-50%) could be localized to A62, A328, and A349+ Combined thioate and c7-deaza interferences were observed at A249 and A330+ Interference effects with average values of ,10% were considered insignificant+ All sites of thioate, c7-deaza, and combined thioate/c7-deaza interference are summarized in the secondary structure model for E. coli RNase P RNA (Fig+ 4)+…”

“…Current three-dimensional models (Chen et al+, 1998;Massire et al+, 1998) represent very valuable but static pictures of the architecture of RNase P RNA-ptRNA complexes, with an estimated resolution of 10-15 Å (Christian et al+, 2000)+ Although we will discuss NAIM results in the context of these models, it should be noted that they do not provide clues with respect to conformational changes of RNase P RNA during its functional cycle or differences in substrate-and productbinding modes (see below)+ Contacts of RNase P RNA to substrate or product RNAs, which are not implied by the current models, may occur at some point in the functional cycle+ Thus, a subset of interference effects may eventually turn out to reflect direct contacts between RNase P RNA and ptRNA or mature tRNA+ The J5/15-P15 region A 6-thioguanine residue at position ϩ1 of the tRNA domain was recently reported to result in a specific short-range crosslink to A248 (Christian et al+, 1998)+ This confirms results of earlier crosslinking studies (Burgin & Pace, 1990; and supports the notion that A248 and A249 are very close to the RNase P cleavage site in E•S and E•P complexes+ C7-deaza interference at A249 was observed in all three NAIM assays (Table 1)+ This interference could be suppressed in the cis-cleavage assay by an increase in the Mg 2ϩ -concentration, suggesting that N7 of A249 may participate in Mg 2ϩ -dependent folding of the active site (Kazantsev & Pace, 1998)+ Precursor tRNA binding was found to be sensitive to methylation of N6 and c7-deaza modification at A248 (Siew et al+, 1999)+ However, a c7-deaza interference at A248 was not seen in the cis-cleavage assay (Table 1, Kazantsev & Pace, 1998)+ We observed a dominant thioate effect at A248, which may have masked a potential c7-deaza tRNA binding interference in our study (Fig+ 3)+ Furthermore, strong thioate interferences at A248 and A249 were only detected in the gel retardation assay (Table 1)+ Such discrepancies suggest that the interference pattern at A248/A249 is sensitive to the functional aspect analyzed, to the mono-and divalent metal ion concentration, and/or the nature of the divalent metal ion present during complex formation (Mg 2ϩ versus Ca 2ϩ )+ In summary, it remains unclear if observed interferences at A248/A249 include effects that represent direct contacts to ptRNA or tRNA, despite the close proximity of A248/A249 to nucleotide ϩ1 of the tRNA domain indicated by the short-range crosslinking data Christian et al+, 1998)+ The same uncertainty pertains to combined thioate/inosine (Heide et al+, 1999) and thioate/c7-deaza interferences at G250/251 (Fig+ 3)+…”

“…Three c7-deaza interferences were observed at the base of or close to P18 (A305, G306, and A328; Fig+ 3)+ Previously, inosine interferences were located to G304, G306, and G329 (Heide et al+, 1999), and 29-deoxy interferences were found at A328 and possibly at U302/ 303 + J18/2 is considered to be part of the catalytic core structure (Harris et al+, 1997;Massire et al+, 1998) and the tetraloop L18 interacts with P8 (Brown et al+, 1996;Harris et al+, 1997;Heide et al+, 1999), thereby supporting the association of domain I and II+ P18 is located on the external or back side in present models of the three-dimensional architecture (Harris et al+, 1997;Chen et al+, 1998;Massire et al+, 1998) and apparently not part of the substrate-or product-binding interface+ However, it should be noted that Kufel and Kirsebom (1996) identified minor crosslinks in P18 with ptRNAs carrying a 4-thiouracil substitution 1 nt upstream of the cleavage site+ Thus, it cannot be excluded that ptRNA or mature tRNA may get in contact with residues in and around P18 at some point of the functional cycle+ Nevertheless, based on current knowledge of the three-dimensional structure of RNase P RNA-ptRNA complexes, the aforementioned interferences more likely identify functional groups supporting the architecture of the active core structure rather than reflecting direct contacts to ptRNA or tRNA+ The extreme sensitivity of the J15/18-P18-J18/2 region towards structural changes was also documented in mutational studies+ For example, the C304/ c7-deaza-modified RNase P RNAsG327 and U321/U328 double mutants showed 10 4 -fold and 10 6 -fold decreases in k cat /K m , respectively + The mutation A317 to G in L18 essentially abolished gel-resolvable tRNA binding, and the corresponding mutant RNase P RNA showed increased susceptibility to Pb 2ϩ -induced hydrolysis in the P15-J15/18-P18 region, indicating that this base exchange causes misfolding (Heide et al+, 1999)+ A strong c7-deaza interference was detected at A334 in all three assays (Table 1)+ In addition, Harris and coworkers found that A334 is sensitive to both deletion and methylation of the N6 amine, implying that A334 may be involved in a Hoogsteen-type hydrogen bonding contact (Siew et al+, 1999)+ In vitro cleavage kinetics and tRNA binding were strongly impaired by A334 to C or U mutations in E. coli RNase P RNA, but not by mutations at the neighboring positions (G332 to C, and A333 to U or C; Baer et al+, 1988;+ This correlates with the relatively low degree of conservation of G332 and A333 compared to A334 among bacterial RNase P RNAs (Haas & Brown, 1998)+ In the presence of ptRNA (with a precursor segment of at least 3-4 nt in length), but not mature tRNA, N1 of A333 showed enhanced susceptibility to DMS methylation, and the N1 and N2 functions of G332 were protected from modification with kethoxal (LaGrandeur et al+, 1994)+ N1 of A334 was inaccessible to DMS methylation in the presence or absence of tRNA or ptRNA, suggesting that adenine 334 is b...…”

“…Here we have applied the nucleotide analog interference mapping (NAIM) approach to study the role of purine N7 positions in the interaction between Escherichia coli RNase P RNA and tRNA+ Previous NAIM studies have identified pro-R p oxygens, 29-OH groups, 2-NH 2 groups of guanines, N7 positions of purines, and 6-NH 2 groups of adenines, whose absence or substitution in E. coli RNase P RNA affects either high-affinity binding of mature tRNA in the presence of Mg 2ϩ (Hardt et al+, 1995bHeide et al+, 1999), cis-cleavage of a ptRNA-RNase P RNA conjugate (Harris & Pace, 1995;Kazantsev & Pace, 1998), or binding of ptRNA in the presence of Ca 2ϩ (Siew et al+, 1999)+ NAIM analyses per se do not permit discrimination between direct or indirect effects as the cause of interference+ Several modifications may affect tRNA binding, ptRNA binding, or the hydrolysis step indirectly by perturbing crucial intramolecular interactions in RNase P RNA or by favoring alternative conformations of the ribozyme+ For example, some interference effects in RNase P RNA that are simultaneously observed in tRNA and ptRNA binding assays under largely different salt conditions (Hardt et al+, 1995bHeide et al+, 1999;Siew et al+, 1999) as well as in the cis-cleavage assay (Harris & Pace, 1995;Kazantsev & Pace, 1998) most likely reflect perturbations of the active core structure of RNase P RNA, which impair mature tRNA binding and ptRNA binding in the ground and transition states+ Additional information has been obtained by comparing interference effects at different Mg 2ϩ concentrations, in the presence of Mn 2ϩ , or by lowering the pH to analyze interference at rate-limiting chemistry (Hardt et al+, 1995b;Harris & Pace, 1995;Kazantsev & Pace, 1998)+ So far, NAIM analyses have identified only two functional groups, the 2-amines of G292 and G293, which unambiguously represent direct contacts between tRNA and RNase P RNA (Heide et al+, 1999)+ Several interference effects could be interpreted in terms of disrupting tertiary interactions in RNase P RNA (L18/P8 and L8/P4; Heide et al+, 1999)+ Other sites of interference were found at or close to residues identified as potential contact sites by photocrosslinking and mutational analyses (Heide et al+, 1999;Siew et al+, 1999), but it is still unclear whether the affected functional groups indeed represent sites of direct contacts to tRNA or ptRNA+…”