Further perspective on the catalytic core and secondary structure of ribonuclease P RNA.

Abstract

Phylogenetic comparative analyses of RNase P RNA-encoding gene sequences from Chlorobium limicola, Chlorobium tepidum, Bacteroides thetaiotaomicron, and Flavobacterium yabuuchiae rerme the secondary structure model of the general (eu)bacterial RNase P RNA and show that a highly conserved feature of that RNA is not essential. Two helices, comprised of 2 base pairs each, are added to the secondary structure model and form part of a cruciform in the RNA. Novel sequence variations in the B. etaiotaomicron and F. yabuuchwae RNA indicate the likelihood that all secondary structure resulting from canonical base-pairing has been detected: there are no remaining unpaired, contiguous, canonical complementarities in the structure model common to all bacterial RNase P RNAs. A nomenclature for the elements of the completed secondary structure model is proposed. The Chlorobium RNase P RNAs lack a stem-oop structure that is otherwise universally present and highly conserved in structure in other (eu)bacterial RNase P RNAs. The Chlorobium RNAs are nevertheless catalytic, with kinetic properties similar to those of RNase P RNAs ofEscherichia coil and other Bacteria. Removal of this stem-oop structure from the E. colt RNA affects neither its affinity for nor its catalytic rate for cleavage of a precursor transfer RNA substrate. These results show that this structural element does not play a direct role in substrate binding or catalysis.RNase P is the site-specific endoribonuclease that removes 5' flanking sequences from precursors of transfer RNA (for reviews, see refs. 1-3). RNase P is a ribonucleoprotein; in Bacteria (formerly eubacteria), in which it is best studied, RNase P is composed of a single ca. 130-kDa RNA and a single ca. 14-kDa protein (see ref. 4 for review). The RNA component of bacterial RNase P is the catalytic moiety; at high ionic strength in vitro it is capable of efficient catalysis in the absence of protein (5). Understanding the mechanisms of substrate recognition and catalysis by RNase P RNA requires knowledge of the structure required for those functions. A model for the secondary structure § of bacterial RNase P RNA has been derived from phylogenetic comparisons (4, 6), which included sequences from representatives of 5 of the approximately 12 "Kingdoms" [sensu Woese (7)] of Bacteria. This secondary structure model was expected to be incomplete; additional sequence variation was required to provide evidence for either the presence or absence of additional structure. Sequences from organisms that are phylogenetically distant from previously examined ones are especially useful for inferring structure because they generally vary greatly from those known. Moreover, phylogenetically disparate sequences may allow the identification of elements in the RNA that are not universally present and, therefore, are presumably not essential for the function ofthe RNA. Naturally occurring gene sequences that lack specific structural elements also provide information useful in designing meaningful deletions or sub...

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

“…Secondary structure of wild-type E. coli RNase P RNA according to Haas et al+ (1994), Mattsson et al+ (1994) and Massire et al+ (1998)+ P: helical regions; L: loop regions; J: joining segments named according to the numbers of helices they connect+ Nucleotides at which an Rp-IMPaS double modification, but not the RpGMPaS modification alone, interfered with tRNA binding are indicated by black circles and white letters; positions where interference effects were enhanced for the Rp-IMPaS compared with the RpGMPaS modification are marked by open circles; circled nucleotides marked by asterisks: because of limited gel resolution, individual contributions to observed interference effects of guanosines at positions G105-109, G250/251 and G259-262 could not be assigned unequivocally+ However, for the latter two regions, main effects appeared to be at G250 and G259/260 (Fig+ 2B)+ The triangle (G68) indicates a 100% Rp-phosphorothioate interference effect+ For the relative strength of interference effects see Table 1+ RNase P RNAs carried an additional G (not shown) at the 59 end for efficient transcription by T7 RNA polymerase+ For RNase P RNAs transcribed from plasmid pSP64M1 linearized with BamHI, the shown 39 end was extended by GGAUC+ In the case of RNase P RNA transcribed from plasmid pSP64M1HH (see Materials and Methods), 39 ends derived from hammerhead self-cleavage had the sequence et al+, 1996)+ The strong inosine interference at G291 is the first biochemical support for a bifurcate H-bonding interaction between the 2-NH 2 group of G291 and the N7 and O6 of G259, as proposed recently in a computer model of of the J15/16 bulge loop complexed with a tRNA ACCA 39-terminus (Easterwood & Harvey, 1997)+ In the aforementioned model, G259, G291, and A 76 of tRNA CCA-termini as well as G292, A258, and C 75 of CCA form coplanar base triples stacked upon each other (Fig+ 7)+ We also identified a c7-deaza modification at G292 that interferes with tRNA binding (unpubl+ results)+ This observation is consistent with H-bonding involving N7 of G292 and the 6-NH 2 group of A258 (Fig+ 7)+ Taken together, our results provide experimental support for several molecular details of the RNase P RNA-NCCA interaction as predicted in the Easterwood and Harvey model+ Further interference effects could be assigned to the region of nt 259-262 (Table 1)+ The four guanosines at this location, however, migrated as two bands on PAA/ urea gels (Fig+ 2B)+ We tentatively assigned one band to G259 and/or G260 and the second to G261 and/or G262+ The interference of intermediate strength was essentially attributable to the Rp-phosphorothioate modification (Table 1)+ The iodine hydrolysis band assigned to G259/260 showed a stronger interference effect than the band assigned to G261/262 (Fig+ 2B)+ G259 of E. coli RNase P RNA was shown to be protected from kethoxal modification (kethoxal modifies the N1 and N2 positions of guanosines) in the presence of (p)tRNA carrying an intact CCA terminus+ Deletion of the terminal A 76 of CCA abolished protection at this location (LaGrandeur et al+, 1994) and A 76 contributes to the high-affinity binding of (p)tRNA to RNase P RNA (Hardt TABLE 1+ Quantification of (A) IMPaS and (B) combined GMPaS/IMPaS interference effects, using a Bio-Imaging Analyzer BAS-1000 (FUJIFILM) and the software PCBAS (Raytest)+ IMPaS: grey-shaded bars; GMPaS: black bars+ Each quantification was based on three to six independent experiments, and deviations between individual experiments were below 6 15%+ To compensate for intensity fluctuations in iodine hydrolysis patterns of tRN...…”

Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA

“…The presence of these conserved sequence elements at hallmark sites in the new genes unambiguously identifies them as templates for nucleus-encoded eukaryal RNase P RNA+ All available eukaryal RNase P RNA sequences were aligned with each other based on conserved elements of primary and secondary RNA structure (this alignment is available at http://pacelab+colorado+edu/ publications+html/)+ The secondary structures of a subset of the RNase P RNA species analyzed are shown in Figure 2+ To generalize the discussion, we designate as "P" (for paired region) those eukaryal helices that are present in all species and are similar in structure and location to bacterial and archaeal counterparts+ These helices are taken to be homologs of the corresponding bacterial and archaeal helices (Haas et al+, 1994)+ Helices designated "eP", for eukaryal paired region, seem for reasons of structure or variable occurrence to be eukaryote specific, not necessarily homologs of bacterial or archaeal helices that might occupy the same position in the universal secondary structure+ Helices eP8 and eP9 may be homologs of the corresponding bacterial and archaeal helices, but the variability in spacing of helices in this region of eukaryal RNase P RNA sequences leaves the homology uncertain+ Table 2 summarizes criteria by which we relate some commonly occurring eukaryal RNase P RNA helices to bacterial and archaeal features+…”

“…a Helix numbering corresponds to the E. coli structure (Haas et al+, 1994)+ b Absent in Methanothermus fervidus (Haas et al+, 1996), Methanococcus jannaschii (Bult et al+, 1996)+ c Absent in Methanococcus jannaschii (Bult et al+, 1996)+ d n+a+: not applicable+ e Homology with bacterial structure uncertain+ FIGURE 2. Secondary structures of eukaryal RNase P RNAs+ Nucleotides that are absolutely conserved in archaeal, bacterial, and eukaryal RNase P RNAs are circled+ The nomenclature for these conserved regions (CRs) is that of Chen and Pace (1997)+ Helices are numbered based on their putative homology to bacterial structures (Haas et al+, 1994)+ Eukaryal helices of uncertain homology to particular bacterial or archaeal structures, but which occur at the same positions in the structures are designated "eP", for eukaryal paired region+ Lower-case nucleotides represent primer sequences+ ( Figure continues on facing page.)…”

“…Secondary structures of eukaryal RNase P RNAs+ Nucleotides that are absolutely conserved in archaeal, bacterial, and eukaryal RNase P RNAs are circled+ The nomenclature for these conserved regions (CRs) is that of Chen and Pace (1997)+ Helices are numbered based on their putative homology to bacterial structures (Haas et al+, 1994)+ Eukaryal helices of uncertain homology to particular bacterial or archaeal structures, but which occur at the same positions in the structures are designated "eP", for eukaryal paired region+ Lower-case nucleotides represent primer sequences+ ( Figure continues on facing page.) Pitulle et al+, 1998) and provide numerous examples of sequence covariations that support the existence of the conserved helices depicted in Figure 2 (i+e+, P3, P4, eP9, P10/11, P12)+ The revised fungal secondary structure models differ from previously proposed fungal RNase P RNA structures (Tranguch & Engelke, 1993) in predicting the existence of helix eP8+ Sequence alignments of both eP8 and eP9 (Fig+ 3) reveal many covarying base pairs that provide evidence for these hairpins+ All fungal eP8 hairpins, with the exception of those of Schizosaccharomyces spp+, contain NUGA tetraloops (the loops of Schizosaccharomyces pombe and Schizosaccharomyces octosporus, in contrast, have the sequence GUGAG)+ With few exceptions, fungal eP9 hairpin loops are of the GNRA type+ In most instances, eP8 is found less than 2 nt 59 of eP9+ Although the spacing between eP8 and eP9 in Saccharomyces dairensis is 23 nt, the sequence alignments unambiguously identify these structures in S. dairensis+ Fungal eP8 and eP9 structures cannot be aligned readily at the primary sequence level with the proposed vertebrate structures+ However, the overall arrangement of helices in the P7/ eP8/eP9/P10/P11 regions of several fungal RNase P RNAs (e+g+, S. pombe, Pichia strasburgensis) is strikingly similar to that of most vertebrate RNase P RNAs (e+g+ Homo sapiens, Danio rerio)+ In these instances, eP8 and eP9, the only helices found in the short span of nucleotides that separates P7 from P10/11, are situated adjacent to P10/P11+ Consequently, we propose that the fungal and vertebrate examples of eP8 and eP9 are homologs, despite the absence of shared sequence identity+ The new sequence data do not support the existence of the previously proposed eukaryal pairing that corresponds to the bacterial helix P5 (Tranguch & Engelke, 1993; Chen & Pace, 1997)+ In bacterial and archaeal RNase P RNAs, helix P5 lies immediately adjacent to the 59 end of helix P4+ The complementarity required to form the corresponding helix generally is absent from the eukaryal sequences+ In H. sapiens, for instance (Fig+ 2), formation of P5 would require pairing of the trinucleotide A86-C87-U88 with G263-C262-G261+ Similarly, formation of P5 would re- quire U-U and G-G appositions in P. strasburgensis (Fig+ 2)+ Like P5, there is no sequence complementarity that would indicate a homolog of the bacterial and archaeal P6+ The apparent absence in the Eukarya of homologs of the bacterial/archaeal helix P5 and P6 may suggest a lack of structural rigidity between the core of eukaryal RNase P RNA (i+e+, helices P1, P2, P3, and P4, the catalytic center of the bacterial RNA) and the remainder of the RNA (i+e+, helices eP7, eP8, eP9, P10/11, and P12)+ In contrast to the known examples of vertebrate RNase P RNA, many of the fungal RNAs contain regions of extensive length variation, particularly in the region between helices eP7 and eP8 (e+g+, Torulaspora delbrueckii, Zygosaccharomyces rouxii )+ In many cases, elements of secondary structure in these regions can be inferred from base complemen...…”

“…Phylogenetic minimum-consensus RNase P RNA secondary structures+ Nucleotides that are absolutely conserved in archaeal, bacterial, and eukaryal RNase P RNAs are circled+ The nomenclature for these conserved regions (CRs) is that of Chen and Pace (1997)+ Helices are numbered based on their putative homology to bacterial structures (Haas et al+, 1994)+ Eukaryal helices of uncertain homology to bacterial or archaeal structures are designated "eP", for eukaryal paired region+ Nucleotides that are invariant within the Eukarya are shown in upper case and those conserved at the 90% level are shown in lower case (R: purine, Y: pyrimidine)+ Nucleotide positions that are found in all eukaryal RNAs, but are not conserved in sequence identity, are shown as black circles+ Arrows represent sites at which one or more phylogenetically variable helices are inserted in selected species+ during their evolution, compared to the bacterial and archaeal versions, yet have retained functionality in the in vivo context, in the presence of protein subunits+…”