Domain structure of the ribozyme from eubacterial ribonuclease P.

Large RNAs can be composed of discrete domains that fold independently. One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA). This domain contains roughly one-third of all residues. Folding of an RNA construct consisting of the remaining two-thirds of B. subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection. This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B. subtilis P RNA. Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+. In order to analyze the structural organization of folding domains in E. coli P RNA, constructs were designed based on the domain structure of B. subtilis P RNA. Fe(II)-EDTA protection indicates that E. coli P RNA also contains two folding domains. Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level. The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other. Because all eubacterial P RNAs share considerable homology in secondary structure to either B. subtilis or E. coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs. Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs.     

Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage

Bacterial ribonuclease P (RNase P) is a ribonucleoprotein complex composed of one catalytic RNA (PRNA) and one protein subunit (P protein) that together catalyze the 5' maturation of precursor tRNA. High-resolution X-ray crystal structures of the individual P protein and PRNA components from several species have been determined, and structural models of the RNase P holoenzyme have been proposed. However, holoenzyme models have been limited by a lack of distance constraints between P protein and PRNA in the holoenzyme-substrate complex. Here, we report the results of extensive cross-linking and affinity cleavage experiments using single-cysteine P protein variants derivatized with either azidophenacyl bromide or 5-iodoacetamido-1,10-o-phenanthroline to determine distance constraints and to model the Bacillus subtilis holoenzyme-substrate complex. These data indicate that the evolutionarily conserved RNR motif of P protein is located near (<15 Angstroms) the pre-tRNA cleavage site, the base of the pre-tRNA acceptor stem and helix P4 of PRNA, the putative active site of the enzyme. In addition, the metal binding loop and N-terminal region of the P protein are proximal to the P3 stem-loop of PRNA. Studies using heterologous holoenzymes composed of covalently modified B. subtilis P protein and Escherichia coli M1 RNA indicate that P protein binds similarly to both RNAs. Together, these data indicate that P protein is positioned close to the RNase P active site and may play a role in organizing the RNase P active site.     

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.     

Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P. Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined. Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives. However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results. Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme. Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor. Further studies showed that the nuclear holoenzyme, but not the B. subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure. The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer. Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition.     

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.     

Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme

The RNA component of bacterial ribonuclease P (RNase P) binds to substrate pre-tRNAs with high affinity and catalyzes site-specific phosphodiester bond hydrolysis to generate the mature tRNA 5' end. Herein we describe the use of biotinylated pre-tRNA substrates to isolate RNase P ribozyme-substrate complexes for nucleotide analogue interference mapping of ribozyme base functional groups involved in substrate recognition. By using a series of adenosine base analogues tagged with phosphorothioate substitutions, we identify specific chemical groups involved in substrate binding. Only 10 adenosines in the Escherichia coli ribozyme show significant sensitivity to interference: A65, A66, A136, A232-234, A248, A249, A334, and A347. Most of these adenosine positions are universally conserved among all bacterial RNase P RNAs; however, not all conserved adenosines are sensitive to analogue substitution. Importantly, all but one of the sensitive nucleotides are located at positions of intermolecular cross-linking between the ribozyme and the substrate. One site of interference that did not correlate with available structural data involved A136 in J11/12. To confirm the generality of the results, we repeated the interference analysis of J11/12 in the Bacillus subtilis RNase P ribozyme, which differs significantly in overall secondary structure. Notably, the B. subtilis ribozyme shows an identical interference pattern at the position (A191) that is homologous to A136. Furthermore, mutation of A136 in the E. coli ribozyme gives rise to a measurable increase in the equilibrium binding constant for the ribozyme-substrate interaction, while mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogue incorporation does not. These results strongly support direct participation of nucleotides in the P4, P11, J5/15, and J18/2 regions of ribozyme structure in pre-tRNA binding and implicate an additional region, J11/12, as involved in substrate recognition. In aggregate, the interference results provide a detailed chemical picture of how the conserved nucleotides adjacent to the pre-tRNA substrate contribute to substrate binding and provide a framework for subsequent identification of the specific roles of these chemical groups in substrate recognition.     

Unstructured RNA is a substrate for tRNase Z

tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.     

Identification of nucleotide residues essential for RNase P activity from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg(2+) ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.     

Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5' maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNA(Asp), highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an Sp phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511-519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.     

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.     

In vitro processing of B. mori transfer RNA precursor molecules.

Ribonuclease P and 3'-5' nuclease, two enzymatic activities necessary for tRNA synthesis in E. coli, are also found in the silkgland cells of Bombyx mori. B. mori subcellular extracts containing RNAase P activity can cleave the E. coli tRNA precursor molecule endonucleolytically at the same site as the E. coli enzyme, and will also cleave in vitro all E. coli tRNA precursors (pre-tRNAs) which the bacterial enzyme recognizes. B. mori RNAase P will not cleave two E. coli RNAase P substrates that are structurally unrelated to tRNA. Pre-tRNAs from B. mori contain extra 5' and 3' nucleotides as judged by RNA fingerprinting and 5' terminal phosphate analysis. Crude silkgland extracts containing both RNAase P and 3'-5' nuclease can remove the 5' and 3' extra nucleotides from B. mori pre-tRNAs, whereas purified fractions containing RNAase P remove only 5' extra nucleotides. Only large silkworm pre-tRNAs were found to be susceptible to cleavage by B. mori RNAase P. This observation and sequence analysis of intermediates of in vitro processing reactions indicate a two-step process of pre-tRNA maturation in which extra 5' nucleotides are first removed by RNAase P and extra 3' nucleotides are then trimmed off by a 3'-5' nuclease.     

Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus.

We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.     

The 5' leader of precursor tRNAAsp bound to the Bacillus subtilis RNase P holoenzyme has an extended conformation

RNase P catalyzes the 5' maturation of transfer RNA (tRNA). RNase P from Bacillus subtilis comprises a large RNA component (130 kDa, P RNA) and a small protein subunit (14 kDa, P protein). Although P RNA alone can efficiently catalyze the maturation reaction in vitro, P protein is strictly required under physiological conditions. We have used time-resolved fluorescence resonance energy transfer on a series of donor-labeled substrates and two acceptor-labeled P proteins to determine the conformation of the pre-tRNA 5' leader relative to the protein in the holoenzyme-pre-tRNA complex. The resulting distance distribution measurements indicate that the leader binds to the holoenzyme in an extended conformation between nucleotides 3 and 7. The conformational mobility of nucleotides 5-8 in the leader is reduced, providing further evidence that these nucleotides interact with the holoenzyme. The increased fluorescence intensity and lifetime of the 5'-fluorescein label of these leaders indicate a more hydrophobic environment, consistent with the notion that such interactions occur with the central cleft of the P protein. Taken together, our data support a model where the P protein binds to the 5' leader between the fourth and seventh nucleotides upstream of the cleavage site, extending the leader and decreasing its structural dynamics. Thus, P protein acts as a wedge to separate the 5' from the 3' terminus of the pre-tRNA and to position the cleavage site in the catalytic core. These results reveal a structural basis for the P protein dependent discrimination between precursor and mature tRNAs.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

The crystal structure of the zinc phosphodiesterase from Escherichia coli provides insight into function and cooperativity of tRNase Z-family proteins

The elaC gene product from Escherichia coli, ZiPD, is a 3' tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3' end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 A. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.     

Molecular analysis of mutated Lactobacillus acidophilus promoter-like sequence P15

The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis. N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L. lactis MG1363. Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T-->A transversion. This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E. coli and Bacillus subtilis vegetative promoters. The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16). The MIC of chloramphenicol in L. lactis, L. reuteri, L. plantarum, E. coli, and L. acidophilus harbouring pLA16 were 30, 170, 180, > 500, and 3 micrograms/mL, respectively. This represents an increase in promoter activity compared to P15 in L. reuteri of 3-fold, in L. plantarum of 9-fold, and in E. coli of at least 2.5-fold, but a decrease in L. acidophilus of 7-fold.