Ribonuclease P

14 Ribonuclease P Sidney Altman Department of Molecular, Cellular and Developmental Biology Yale University New Haven, Connecticut 06520 Leif Kirsebom Department of Microbiology Biomedical Center Uppsala University Uppsala S-751 23, Sweden RNase P was first characterized in Escherichia coli as an enzyme that is required for the processing of the 5؅ termini of tRNA in the pathway of biosynthesis of tRNA from precursor tRNAs (ptRNAs; Altman and Smith 1971; for review, see Altman 1989). Subsequently, it was discovered that this enzyme consisted of one protein and one RNA subunit, the latter being the catalytic subunit (Guerrier-Takada et al. 1983). In retro- spect, it should not be surprising that an enzyme with the chemical compo- sition of RNase P exists. Ribosomes are also ribonucleoproteins (RNPs), but they are much more complicated than RNase P. It seems unreasonable that ribosomes, with three RNAs and about 50 proteins, would exist without less complex RNPs having come into existence first and having per- sisted throughout evolution. Indeed, relatively simple RNPs with and without catalytic activity have been discovered with regularity during the past 20 years. What important questions about RNase P have not yet been an- swered? Certainly, for the biochemist, problems not yet solved for E. coli RNase P include a full understanding of enzyme–substrate interactions, RNA–protein subunit interactions, and the chemical details of the hydrolysis reaction. In every case, much progress has been made, but much has yet to be learned. A second issue of interest to biochemists and “evolutionists” is the puzzle presented by the different compositions of RNase P from eubac- teria (one catalytic RNA and one protein subunit) and from eukaryotes (one RNA subunit and several protein subunits: no understanding yet of which subunit is responsible for catalysis). The complexity of eukaryotic The RNA World, Second Edition © 1999 Cold Spring Harbor Laboratory Press 0-87969-561-7/99 351 352 S. Altman and L.A. Kirsebom RNase P presents the questions of why there are so many subunits and what relation this complexity might have to the intracellular localization of the enzyme, possible isoforms (Li and Williams 1995), and its relation to other enzymes with which it might interact in vivo. RNase P in orga- nelles, e.g., chloroplasts and mitochondria, present their own complexities in terms of the origin and function of their subunits (Wang et al. 1988; Dang and Martin 1993; Baum et al. 1996; Lang et al. 1997; Martin and Lang 1997). More generally, the role of RNase P in the regulation of tRNA and rRNA biosynthesis in both prokaryotes and eukaryotes has not yet been fully explored. Indeed, new roles for RNase P in cell physiology are still being uncovered (Stolc and Altman 1997). This chapter focuses on enzyme–substrate interactions and summa- rizes briefly what is known about RNase P from bacteria (predominantly E. coli) that is pertinent to the other questions presented above and con- trasts the characteristics of E. coli RNase P with eukaryotic RNase P (predominantly human RNase P). The discussion is not meant to be encyclo- pedic: We apologize to those whose work we have not mentioned. FUNCTION OF RNASE P In E. coli, RNase P processes not only ptRNAs (Fig. 1) but also, at the very least, the precursors to 4.5S RNA (Bothwell et al. 1976a), an analog of the eukaryotic SRP RNA (Poritz et al. 1990), tmRNA (10Sa RNA; Komine et al. 1994; Keiler et al. 1996; Muto et al. 1996; Felden et al. 1997), and some small phage RNAs (Hartmann et al. 1995). Formally, this enzyme also has another substrate, 30S prRNA, which it cleaves at the 5؅ termini of tRNA sequences embedded in the long prRNA (for reviews, see Apirion and Miczak 1993; Deutscher 1993). There may very well be other “hidden” substrates, e.g., ones that resemble the polycistronic his operon mRNA, which have not yet been identified in the complexity of RNA processing events in vivo. In this latter case, a cleavage site by RNase P is accessible only after a previous cleavage of the mRNA by RNase E (Alifano et al. 1994). Only after the RNase E cleavage can the -remaining 3؅ terminal fragment of the polycistronic mRNA adopt a con formation that presents the recognition features of substrates for RNase P. To date, only one set of substrates for purified human RNase P has been identified: These are ptRNAs. However, in crude extracts of both yeast and HeLa cells, RNase P appears to be part of a complex that includes RNase MRP and that is involved in the cleavage of prRNAs (Lygerou et al. 1996; Jarrous et al. 1998). Highly purified RNase P, however, cannot carry out this latter cleavage reaction (Jarrous et al. 1998). Ribonuclease P 353 Figure 1 Natural substrates for RNase P from E. coli. Secondary structures of several substrates are shown. (A) Precursor to tRNATyr. (B) Precursor to 4.5S RNA. (C) Precursor to tmRNA. (D) Precursor to phages P1 and P7 antisense C4 RNA. (E) his operon mRNA at the junction of the his C and his B genes of Salmonella typhimurium (see text for explanation and references). Identification of new substrates for eukaryotic RNase P should involve the examination of cell extracts depleted in RNase P activity. The fact that nuclear RNase P from both human and yeast cells have several protein subunits points to the possibility that these subunits, alone or together in the RNase P holoenzyme complex, have a function other than that of RNA cleavage. (RNase P from Saccharomyces cerevisiae and HeLa cells [Eder et al. 1997; Chamberlain et al. 1998] have been suffi- ciently highly purified to enable the previous statement regarding the number of subunits to be made. In S. cerevisiae, there is also genetic evi- dence for the identity of at least five subunits [for references, see Stolc and Altman 1997; Stolc et al. 1998].) In fact, the sequences of at least two of the human subunits have motifs resembling those found in proteins that are known to have nuclear localization signals (Jarrous et al. 1998). 354 S. Altman and L.A. Kirsebom In human cells, RNase P must also be transported to the mitochondria (Doersen et al. 1985). (Unlike mitochondria in human cells, in several yeasts and other lower eukaryotes, the mitochondria appear to encode their own RNase P [Hollingsworth and Martin 1986; Martin and Lang 1997].) Accordingly, one of the protein subunits of the human enzyme may be important for the transport process. One candidate is the protein called p38 or Rpp38 (Eder et al. 1997), a nuclear antigen that is also found in the RNP enzyme known as RNase MRP (Yuan et al. 1991). As with RNase P, this enzyme is thought to have both nuclear (nucleolar) and mitochondrial function. The p38-binding domains in the RNA subunits of each of these enzymes are similar in structure (see Fig. 2) (Forster and Altman 1990b). Indeed, there are significant similarities in the overall proposed secondary structure of these RNAs. We note also that the two enzymatic functions are physically associated with each other in crude extracts of both human and yeast cells (Tollervey 1995; Stolc and Altman 1997), an indication of function in similar, or the same, biosynthetic path- ways (e.g., rRNA biosynthesis) and perhaps a common origin during evolution (see below). What are the common features of the various substrates that are recognized by RNase P? Extensive genetic and biochemical analyses of both natural and model substrates indicate that the minimal features recognized by E. coli RNase P are a hydrogen-bonded stem of >3 bp and at least one .(unpaired nucleotide at the 5؅ end of the sequence (Liu and Altman 1996 The sequence –CCA at the 3؅ end of this stem (common to all mature tRNAs) enhances cleavage efficiency (Seidman and McClain 1975; Guerrier-Takada et al. 1984; Green and Vold 1988). The variety of known model substrates is shown in Figure 3. In contrast, human RNase P requires a somewhat more complex minimal structure for recognition by the enzyme (Fig. 3), one in which there is a bulge of one or more nucleotides at position 8 (Yuan and Altman 1995; see below). SOME DETAILS OF THE REACTION WITH DIFFERENT SUBSTRATES The simplest way of studying an enzymatic reaction is to perform an analysis of its kinetics and to quantitate the well-known parameters, Km and kcat. The values of these parameters for several substrates for E. coli RNase P are listed in Table 1. Note that whereas some model substrates have a value of kcat/Km (a measure of the efficiency of the reaction) close to that of the enzyme with a natural substrate, the Km with ptRNA is the lowest, an indication that the interaction between ptRNA and the holoenzyme is stronger than with other substrates. We conclude, not Ribonuclease P Figure 2 Secondary structure schemes of (A) M1 RNA and (B) H1 RNA. The domain of H1 RNA that is analogous to a domain to which the Th/To antigen, p38, binds (Yuan et al. 1991) in MRP RNA is shown in bold letters (Forster and Altman 1990b). (A, 355 Reprinted, with permission, from Chen and Pace 1997, B modified from Chen and Pace 1997.) 356 S. Altman and L.A. Kirsebom unexpectedly, that a natural substrate has more contacts with the enzyme than do model substrates. Indeed, mutagenesis studies show that any mutation that disturbs the secondary and tertiary structure of the tRNA moiety of the substrate in any part of the molecule alters the Km of the Figure 3 Model substrates for E.

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