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Catalysis by the RNA subunit of RNase P — a minireview
63
Gene, 82 (1989) 63-64 Elsevier GENE
03146
Catalysis by the RNA subunit of RNase P -
a minireview *
(Catalytic RNA; model substrates; Ml RNA structure and function; protein cofactor; nucleotide sequences)
S. Altman, M.F. Baer, M. Bartkiewicz, H. Gold, C. Guerrier-Takada, L.A. Kirsebom, N. Lumelsky and K. Peck Department of Biology, Yale University,New Haven, CT 06S20 (U.S.A.) Received
by M. Belfort:
Accepted:
27 February
5 October
1988
1989
SUMMARY
RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5’ termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.
INTRODUCTION
RNase P is an enzyme that cleaves tRNA precursor molecules to generate the 5’ termini of mature tRNAs (reviewed in Altman et al., 1986; Lawrence et al., 1987a; Altman, 1989). An RNase P-like activity has been found in extracts of all organisms thus far examined. RNase P from several organisms has been shown to contain both essential RNA and protein components. The RNA subunits from some prokaryotes have been prepared by transcription in vitro and manifest catalytic activity and all the other properties of a classical enzyme. Correspondenceto: Dr. S. Altman, University,
New Haven,
Department
of Biology, Yale
CT 06520 (U.S.A.) Tel. (203)432-3506;
Fax (203)432-6161. * Presented Splicing, tember
at the Albany
Evolution’,
Conference
Rensselaerville,
on ‘RNA:
NY (U.S.A.)
Catalysis, 22-25
Sep-
1988.
Abbreviations: component ribosomal
bp, base of RNase
pair(s);
Ml
RNA,
a catalytic
P from E. coli; nt, nucleotide(s);
RNA; ss, single strand(ed);
tRNA, transfer
RNA rRNA,
RNA; wt,
wild type.
0378-l 119/89/$03.50
0 1989 Elsevier
Science Publishers
B.V. (Biomedical
Our studies concern mainly Ml RNA, the RNA subunit of RNase P from Escherichia co/i. We have studied model substrates for this enzyme, as well as a variety of deletion and point mutants of the enzyme itself, to gain more insight into the mechanism of action of this catalytic RNA and into the ancient past of catalytic RNA.
PROPERTIES
OF THE ENZYME
SYSTEM
(a) Substrates
Simple substrates consisting of a short 5’ ss region followed by a stem-loop structure and ending in CCA, can be cleaved by Ml RNA or the holoenzyme complex (McClain et al., 1987). As few as two (and possibly one) nt are required in the 5’ ss region and six (or possibly fewer) bp are needed in the stem region of the substrates. Alterations in the CCA sequence can lead to a drastic reduction in the cleavage rate of these substrates, by Ml RNA. In some cases, the RNase P holoenzyme complex can Division)
64
overcome the effect of altering the CCA sequence. While these and other similar small analogs (Guerrier-Takada et al., 1988) of tRNA precursors can be cleaved in vitro, it is not apparent that they function as substrates in vivo. The context in which these structures are embedded in larger RNA molecules determines whether or not they are substrates in vivo. However, these small substrates may represent the simplest substrate (proto-substrate) for M 1 RNA that existed eons ago.
Ml RNA and 23s rRNA (D. Moazed, J. Robertson and H. Noller, personal communication) bind deacylated tRNA. We have identified similarities in sequence and structure in regions of Ml RNA and 23s rRNA that are found to be in intimate contact with tRNA. We have not yet determined the degree of functional similarity between these two sites or what role the protein cofactor of RNase P plays in the binding of tRNA at this site.
(b) The enzyme
ACKNOWLEDGEMENTS
The first models for the secondary structure of M 1 RNA were based on theoretical considerations of helix stabilities, susceptibility of the molecule to nucleases in solution and limited sequence comparisons (Lawrence et al., 1987b). A more recent, improved model is based on more extensive phylogenetic comparison (James et al., 1988). However, the results of extensive analysis of mutants of M 1 RNA (Lumelsky and Altman, 1988) are not totally consistent with the current models and the limited speculation conceming the tertiary interactions in Ml RNA that they contain. A model for the analog of Ml RNA from HeLa cells does bear a striking similarity to the one proposed for prokaryotes, even though the sequences of these RNAs have diverged widely through evolutionary time (Bartkiewicz et al., 1989). An analysis of large deletion mutants of Ml RNA indicates that two regions (widely separated in the primary structure) are critical for enzymatic activity. They lie between nt 20 and 95 and nt 205 and 255. A large deletion lacking nt 15-205 can cleave a small model substrate, but not a wt tRNA precursor. This small derivative of Ml RNA may represent a proto-enzyme that has the capability of cleaving a proto-substrate.
Work in the laboratory of S.A. is supported by grants from the USPHS and USNSF. K.P. is the recipient of a predoctoral training award. N.L. is the recipient of a postdoctoral fellowship from the USPHS.
(c) A tRNA-binding site in Ml RNA?
Both Ml RNA and 23s rRNA are found in vivo in ribonucleoprotein particles. The protein cofactor of RNase P affects the rate of cleavage reaction and interacts in a specific manner with different precursor tRNAs, although the nature of this interaction is not fully understood (Kirsebom et al., 1988). The many proteins in the large ribosomal subunit are involved in several functions of the ribosome. Both
REFERENCES Altman, S.: Ribonuclease P: an enzyme with a catalytic RNA subunit. Adv. Enzymol. 62 (1989) l-36. Altman, S., Baer, M., Guerrier-Takada, C. and Vioque, A.: Enzymatic cleavage of RNA by RNA. Trends Biochem. Sci. 11 (1986) 515-518. Bartkiewicz, M., Gold, H. and Altman, S.: Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells. Genes Develop. 3 (1989) 488-499. Guerrier-Takada, C., Van Belkum, A., Pleij, C.W.A. and Altman, S.: Novel reactions of RNase P with a tRNA-like structure in turnip yellow mosaic virus RNA. Cell 53 (1988) 267-272. James, B.D., Olsen, G.J. and Pace, N.R.: The secondary structure ofribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme. Cell 52 (1988) 19-26. Kirsebom, L.A., Baer, M.F. and Altman, S.: Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors. J. Mol. Biol. 204 (1988) 879-888. Lawrence, N., Wesolowski, D., Gold, H., Bartkiewicz, M., Guerrier-Takada, C., McClain, W.H. and Altman, S.: Characteristics of ribonuclease P from various organisms. Cold Spring Harbor Symp. Quant. Biol. 52 (1987a) 233-238. Lawrence, N.P., Richman, A., Amini, R. and Altman, S.: Heterologous enzyme function in Escherichiu coli and the selection of genes encoding the catalytic RNA subunit of RNase P. Proc. Natl. Acad. Sci. USA 84 (1987b) 6825-6829. Lumelsky, N. and Altman, S.: Selection and characterization of randomly produced mutants in the gene coding for M 1 RNA. J. Mol. Biol. 202 (1988) 443-454. McClain, W.H., Guerrier-Takada, C. and Altman, S.: Model substrates for an RNA enzyme. Science 238 (1987) 527-530.
Gene, 82 (1989) 63-64 Elsevier GENE
03146
Catalysis by the RNA subunit of RNase P -
a minireview *
(Catalytic RNA; model substrates; Ml RNA structure and function; protein cofactor; nucleotide sequences)
S. Altman, M.F. Baer, M. Bartkiewicz, H. Gold, C. Guerrier-Takada, L.A. Kirsebom, N. Lumelsky and K. Peck Department of Biology, Yale University,New Haven, CT 06S20 (U.S.A.) Received
by M. Belfort:
Accepted:
27 February
5 October
1988
1989
SUMMARY
RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5’ termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.
INTRODUCTION
RNase P is an enzyme that cleaves tRNA precursor molecules to generate the 5’ termini of mature tRNAs (reviewed in Altman et al., 1986; Lawrence et al., 1987a; Altman, 1989). An RNase P-like activity has been found in extracts of all organisms thus far examined. RNase P from several organisms has been shown to contain both essential RNA and protein components. The RNA subunits from some prokaryotes have been prepared by transcription in vitro and manifest catalytic activity and all the other properties of a classical enzyme. Correspondenceto: Dr. S. Altman, University,
New Haven,
Department
of Biology, Yale
CT 06520 (U.S.A.) Tel. (203)432-3506;
Fax (203)432-6161. * Presented Splicing, tember
at the Albany
Evolution’,
Conference
Rensselaerville,
on ‘RNA:
NY (U.S.A.)
Catalysis, 22-25
Sep-
1988.
Abbreviations: component ribosomal
bp, base of RNase
pair(s);
Ml
RNA,
a catalytic
P from E. coli; nt, nucleotide(s);
RNA; ss, single strand(ed);
tRNA, transfer
RNA rRNA,
RNA; wt,
wild type.
0378-l 119/89/$03.50
0 1989 Elsevier
Science Publishers
B.V. (Biomedical
Our studies concern mainly Ml RNA, the RNA subunit of RNase P from Escherichia co/i. We have studied model substrates for this enzyme, as well as a variety of deletion and point mutants of the enzyme itself, to gain more insight into the mechanism of action of this catalytic RNA and into the ancient past of catalytic RNA.
PROPERTIES
OF THE ENZYME
SYSTEM
(a) Substrates
Simple substrates consisting of a short 5’ ss region followed by a stem-loop structure and ending in CCA, can be cleaved by Ml RNA or the holoenzyme complex (McClain et al., 1987). As few as two (and possibly one) nt are required in the 5’ ss region and six (or possibly fewer) bp are needed in the stem region of the substrates. Alterations in the CCA sequence can lead to a drastic reduction in the cleavage rate of these substrates, by Ml RNA. In some cases, the RNase P holoenzyme complex can Division)
64
overcome the effect of altering the CCA sequence. While these and other similar small analogs (Guerrier-Takada et al., 1988) of tRNA precursors can be cleaved in vitro, it is not apparent that they function as substrates in vivo. The context in which these structures are embedded in larger RNA molecules determines whether or not they are substrates in vivo. However, these small substrates may represent the simplest substrate (proto-substrate) for M 1 RNA that existed eons ago.
Ml RNA and 23s rRNA (D. Moazed, J. Robertson and H. Noller, personal communication) bind deacylated tRNA. We have identified similarities in sequence and structure in regions of Ml RNA and 23s rRNA that are found to be in intimate contact with tRNA. We have not yet determined the degree of functional similarity between these two sites or what role the protein cofactor of RNase P plays in the binding of tRNA at this site.
(b) The enzyme
ACKNOWLEDGEMENTS
The first models for the secondary structure of M 1 RNA were based on theoretical considerations of helix stabilities, susceptibility of the molecule to nucleases in solution and limited sequence comparisons (Lawrence et al., 1987b). A more recent, improved model is based on more extensive phylogenetic comparison (James et al., 1988). However, the results of extensive analysis of mutants of M 1 RNA (Lumelsky and Altman, 1988) are not totally consistent with the current models and the limited speculation conceming the tertiary interactions in Ml RNA that they contain. A model for the analog of Ml RNA from HeLa cells does bear a striking similarity to the one proposed for prokaryotes, even though the sequences of these RNAs have diverged widely through evolutionary time (Bartkiewicz et al., 1989). An analysis of large deletion mutants of Ml RNA indicates that two regions (widely separated in the primary structure) are critical for enzymatic activity. They lie between nt 20 and 95 and nt 205 and 255. A large deletion lacking nt 15-205 can cleave a small model substrate, but not a wt tRNA precursor. This small derivative of Ml RNA may represent a proto-enzyme that has the capability of cleaving a proto-substrate.
Work in the laboratory of S.A. is supported by grants from the USPHS and USNSF. K.P. is the recipient of a predoctoral training award. N.L. is the recipient of a postdoctoral fellowship from the USPHS.
(c) A tRNA-binding site in Ml RNA?
Both Ml RNA and 23s rRNA are found in vivo in ribonucleoprotein particles. The protein cofactor of RNase P affects the rate of cleavage reaction and interacts in a specific manner with different precursor tRNAs, although the nature of this interaction is not fully understood (Kirsebom et al., 1988). The many proteins in the large ribosomal subunit are involved in several functions of the ribosome. Both
REFERENCES Altman, S.: Ribonuclease P: an enzyme with a catalytic RNA subunit. Adv. Enzymol. 62 (1989) l-36. Altman, S., Baer, M., Guerrier-Takada, C. and Vioque, A.: Enzymatic cleavage of RNA by RNA. Trends Biochem. Sci. 11 (1986) 515-518. Bartkiewicz, M., Gold, H. and Altman, S.: Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells. Genes Develop. 3 (1989) 488-499. Guerrier-Takada, C., Van Belkum, A., Pleij, C.W.A. and Altman, S.: Novel reactions of RNase P with a tRNA-like structure in turnip yellow mosaic virus RNA. Cell 53 (1988) 267-272. James, B.D., Olsen, G.J. and Pace, N.R.: The secondary structure ofribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme. Cell 52 (1988) 19-26. Kirsebom, L.A., Baer, M.F. and Altman, S.: Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors. J. Mol. Biol. 204 (1988) 879-888. Lawrence, N., Wesolowski, D., Gold, H., Bartkiewicz, M., Guerrier-Takada, C., McClain, W.H. and Altman, S.: Characteristics of ribonuclease P from various organisms. Cold Spring Harbor Symp. Quant. Biol. 52 (1987a) 233-238. Lawrence, N.P., Richman, A., Amini, R. and Altman, S.: Heterologous enzyme function in Escherichiu coli and the selection of genes encoding the catalytic RNA subunit of RNase P. Proc. Natl. Acad. Sci. USA 84 (1987b) 6825-6829. Lumelsky, N. and Altman, S.: Selection and characterization of randomly produced mutants in the gene coding for M 1 RNA. J. Mol. Biol. 202 (1988) 443-454. McClain, W.H., Guerrier-Takada, C. and Altman, S.: Model substrates for an RNA enzyme. Science 238 (1987) 527-530.