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concentrations will affect the proportion of charging of G3:U70 tRNA T~ with alanine and tyrosine, the extent of these changes depends entirely on the parameters for the cross-interactions. These additional interactions cannot be factored into the calculations without more experimental data. However, were such parameters available, the numerical methods that have been used here can accommodate additional reactions by a straightforward generalization of the procedures that are outlined here. Acknowledgment This work was supported by Grant GM 23562 from the National Institutes of Health (NIH). J. J. B. holds NIH postdoctoral fellowship GM 12122.
[26]
Design of Simplified Ribonuclease P RNA by Phylogenetic Comparison By NORMAN R. PACE a n d DAVID S. WAUGH
Introduction Complex biological macromolecules commonly are multifunctional and consist of discrete structural domains with different functions. The fabrication of abbreviated versions of such complex molecules allows study or use of a particular function in isolation from the rest of the natural molecule. The design and testing of simplified enzymes is also an approach to identifying elements of molecular structure required for catalysis. The goal is to construct the smallest active version of an enzyme and thereby to determine the minimum structure required for catalysis. We have used this approach to outline the catalytic core of the RNA enzyme ribonuclease P (RNase p).l RNase P, an endonuclease, forms the 5' ends of tRNAs. In eubacteria RNase P consists of essential protein (ca. 14 kDa) and RNA (ca. 400 nucleotides) components,z,3 However, the RNase P RNA alone is an efficient and accurate catalyst at high salt concentrations in vitro. The high ionic strength evidently screens anionic repulsion between the enzyme and substrate RNAs. In order to identify potentially important structure in D. S. Waugh, C. J. Green, and N. R. Pace, Science 244, 1569 (1989). 2 S. Airman, Adv. Enzymol. Relat. AreasMol. Biol. 62, 1 (1989). 3 N. R. Pace and D. Smith, J. Biol. Chem. 265, 3587 (1990).
METHODS IN ENZYMOLOGY, VOL. 203
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[26]
DESIGN OF SIMPLIFIED RIBONUCLEASEP R N A
501
RNase P RNA, we undertook to delete sequences that are not required for activity, seeking the minimum functional structure. This approach has been useful in localizing the portion of tRNA that is recognized by RNase p4 and in the study of other catalytic RNAs. 5,6 Design of Min 1 R N A The RNase P RNAs are too complex for an eti~cient identification of nonessential sequences by random deletion mutagenesis. We therefore used a phylogenetic comparison of secondary structure models to identify potentially dispensable sequences. Secondary structure models of nine RNase P RNAs from two eubacterial phyla, the gram-positive bacteria (e.g., Bacillus megaterium in Fig. 1) and the "purple bacteria," (e.g., Escherichia coli in Fig. 1), have been derived using a comparative approach to identify base-paired sequences.7 Differences between the models of the RNAs from the two phyla are due substantially to the phylum-specific occurrence of discrete helical elements at various positions in a core of homologous primary and secondary structure. 7,8 Since the phylum-specific helical elements are not present in the RNase P RNAs of all organisms, they are not expected to be crucial for the enzymatic activity of the RNA. We relied on the structure models to design a simplified RNase P RNA that consists only of phylogenetically conserved features. The E. coli RNase P RNA (M1 RNA) was used as the foundation of the design because its structure model has fewer interruptions in the conserved core than do those of the RNase P RNAs from gram-positive bacteria. There are four proposed helices in the E. coli RNase P RNA model that have no counterparts in the gram-positive versions and so are not expected to be essential for enzymatic activity. These helices involve nucleotides 28-52, 148- 167, 183-227, and 260-290 of the folded E. coli sequence in Fig. 1. They were excluded from the design of the simplified RNA using two strategies: simple omission from the design, and replacement ofE. coli sequences with homologous segments of a gram-positive P RNA that lacks the E. col#specific helical elements. Nucleotides 28-52 in the E. coli RNase P RNA structure model form the apical part of a longer helix that involves nucleotides 20- 61. Since only 4 W. H. McClain, C. Guerrier-Takada, and S. Altman, Science 23, 527 (1987). s j. W. Szostak, Nature (London) 322, 83 (1986). 60. C. Uhlenbeck, Nature (London) 328, 596 (1987). 7 B. D. Jones, G. J. Olsen, J. Liu, and N. R. Pace, Cell (Cambridge, Mass.) 52, 19 (1988). 8 We use the term homologous in its strictest sense: homologous sequences have common ancestry and function. Homologous sequences are not necessarily identical; identical sequences are not necessarily homologous.
502
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[26]
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[26]
concentrations will affect the proportion of charging of G3:U70 tRNA T~ with alanine and tyrosine, the extent of these changes depends entirely on the parameters for the cross-interactions. These additional interactions cannot be factored into the calculations without more experimental data. However, were such parameters available, the numerical methods that have been used here can accommodate additional reactions by a straightforward generalization of the procedures that are outlined here. Acknowledgment This work was supported by Grant GM 23562 from the National Institutes of Health (NIH). J. J. B. holds NIH postdoctoral fellowship GM 12122.
[26]
Design of Simplified Ribonuclease P RNA by Phylogenetic Comparison By NORMAN R. PACE a n d DAVID S. WAUGH
Introduction Complex biological macromolecules commonly are multifunctional and consist of discrete structural domains with different functions. The fabrication of abbreviated versions of such complex molecules allows study or use of a particular function in isolation from the rest of the natural molecule. The design and testing of simplified enzymes is also an approach to identifying elements of molecular structure required for catalysis. The goal is to construct the smallest active version of an enzyme and thereby to determine the minimum structure required for catalysis. We have used this approach to outline the catalytic core of the RNA enzyme ribonuclease P (RNase p).l RNase P, an endonuclease, forms the 5' ends of tRNAs. In eubacteria RNase P consists of essential protein (ca. 14 kDa) and RNA (ca. 400 nucleotides) components,z,3 However, the RNase P RNA alone is an efficient and accurate catalyst at high salt concentrations in vitro. The high ionic strength evidently screens anionic repulsion between the enzyme and substrate RNAs. In order to identify potentially important structure in D. S. Waugh, C. J. Green, and N. R. Pace, Science 244, 1569 (1989). 2 S. Airman, Adv. Enzymol. Relat. AreasMol. Biol. 62, 1 (1989). 3 N. R. Pace and D. Smith, J. Biol. Chem. 265, 3587 (1990).
METHODS IN ENZYMOLOGY, VOL. 203
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[26]
DESIGN OF SIMPLIFIED RIBONUCLEASEP R N A
501
RNase P RNA, we undertook to delete sequences that are not required for activity, seeking the minimum functional structure. This approach has been useful in localizing the portion of tRNA that is recognized by RNase p4 and in the study of other catalytic RNAs. 5,6 Design of Min 1 R N A The RNase P RNAs are too complex for an eti~cient identification of nonessential sequences by random deletion mutagenesis. We therefore used a phylogenetic comparison of secondary structure models to identify potentially dispensable sequences. Secondary structure models of nine RNase P RNAs from two eubacterial phyla, the gram-positive bacteria (e.g., Bacillus megaterium in Fig. 1) and the "purple bacteria," (e.g., Escherichia coli in Fig. 1), have been derived using a comparative approach to identify base-paired sequences.7 Differences between the models of the RNAs from the two phyla are due substantially to the phylum-specific occurrence of discrete helical elements at various positions in a core of homologous primary and secondary structure. 7,8 Since the phylum-specific helical elements are not present in the RNase P RNAs of all organisms, they are not expected to be crucial for the enzymatic activity of the RNA. We relied on the structure models to design a simplified RNase P RNA that consists only of phylogenetically conserved features. The E. coli RNase P RNA (M1 RNA) was used as the foundation of the design because its structure model has fewer interruptions in the conserved core than do those of the RNase P RNAs from gram-positive bacteria. There are four proposed helices in the E. coli RNase P RNA model that have no counterparts in the gram-positive versions and so are not expected to be essential for enzymatic activity. These helices involve nucleotides 28-52, 148- 167, 183-227, and 260-290 of the folded E. coli sequence in Fig. 1. They were excluded from the design of the simplified RNA using two strategies: simple omission from the design, and replacement ofE. coli sequences with homologous segments of a gram-positive P RNA that lacks the E. col#specific helical elements. Nucleotides 28-52 in the E. coli RNase P RNA structure model form the apical part of a longer helix that involves nucleotides 20- 61. Since only 4 W. H. McClain, C. Guerrier-Takada, and S. Altman, Science 23, 527 (1987). s j. W. Szostak, Nature (London) 322, 83 (1986). 60. C. Uhlenbeck, Nature (London) 328, 596 (1987). 7 B. D. Jones, G. J. Olsen, J. Liu, and N. R. Pace, Cell (Cambridge, Mass.) 52, 19 (1988). 8 We use the term homologous in its strictest sense: homologous sequences have common ancestry and function. Homologous sequences are not necessarily identical; identical sequences are not necessarily homologous.
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NUCLEIC ACIDS AND POLYSACCHARIDES
[26]
