Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Oct;77(10):5644–5648. doi: 10.1073/pnas.77.10.5644

Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

D A Stahl, B Meyhack, N R Pace
PMCID: PMC350125  PMID: 6777771

Abstract

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.

Full text

PDF
5644

Images in this article

5646Image
on p.5646
5646Image
on p.5646
5647Image
on p.5647
5647Image
on p.5647
5647Image
on p.5647

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abelson J. RNA processing and the intervening sequence problem. Annu Rev Biochem. 1979;48:1035–1069. doi: 10.1146/annurev.bi.48.070179.005131. [DOI] [PubMed] [Google Scholar]
  2. Arnott S., Chandrasekaran R., Leslie A. G. Structure of the single-stranded polyribonucleotide polycytidylic acid. J Mol Biol. 1976 Sep 25;106(3):735–748. doi: 10.1016/0022-2836(76)90262-x. [DOI] [PubMed] [Google Scholar]
  3. Barrio J. R., Barrio M. C., Leonard N. J., England T. E., Uhlenbeck O. C. Synthesis of modified nucleoside 3',5'-bisphosphates and their incorporation into oligoribonucleotides with T4 RNA ligase. Biochemistry. 1978 May 30;17(11):2077–2081. doi: 10.1021/bi00604a009. [DOI] [PubMed] [Google Scholar]
  4. Cameron V., Uhlenbeck O. C. 3'-Phosphatase activity in T4 polynucleotide kinase. Biochemistry. 1977 Nov 15;16(23):5120–5126. doi: 10.1021/bi00642a027. [DOI] [PubMed] [Google Scholar]
  5. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. England T. E., Uhlenbeck O. C. Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. Biochemistry. 1978 May 30;17(11):2069–2076. doi: 10.1021/bi00604a008. [DOI] [PubMed] [Google Scholar]
  7. Fox G. E., Woese C. R. 5S RNA secondary structure. Nature. 1975 Aug 7;256(5517):505–507. doi: 10.1038/256505a0. [DOI] [PubMed] [Google Scholar]
  8. Kallenbach N. R., Berman H. M. RNA structure. Q Rev Biophys. 1977 May;10(2):138–236. doi: 10.1017/s0033583500000202. [DOI] [PubMed] [Google Scholar]
  9. Meyhack B., Pace B., Pace N. R. Involvement of precursor-specific segments in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA. Biochemistry. 1977 Nov 15;16(23):5009–5015. doi: 10.1021/bi00642a011. [DOI] [PubMed] [Google Scholar]
  10. Meyhack B., Pace B., Uhlenbeck O. C., Pace N. R. Use of T4 RNA ligase to construct model substrates for a ribosomal RNA maturation endonuclease. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3045–3049. doi: 10.1073/pnas.75.7.3045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyhack B., Pace N. R. Involvement of the mature domain in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA. Biochemistry. 1978 Dec 26;17(26):5804–5810. doi: 10.1021/bi00619a030. [DOI] [PubMed] [Google Scholar]
  12. Mizuno H., Sundaralingam M. Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction. Nucleic Acids Res. 1978 Nov;5(11):4451–4461. doi: 10.1093/nar/5.11.4451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schimmel P. R., Söll D. Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem. 1979;48:601–648. doi: 10.1146/annurev.bi.48.070179.003125. [DOI] [PubMed] [Google Scholar]
  14. Sninsky J. J., Last J. A., Gilham P. T. The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules. Nucleic Acids Res. 1976 Nov;3(11):3157–3166. doi: 10.1093/nar/3.11.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sogin M. L., Pace B., Pace N. R. Partial purification and properties of a ribosomal RNA maturation endonuclease from Bacillus subtilis. J Biol Chem. 1977 Feb 25;252(4):1350–1357. [PubMed] [Google Scholar]
  16. Sogin M. L., Pace N. R. Nucleotide sequence of 5 S ribosomal RNA precursor from Bacillus subtilis. J Biol Chem. 1976 Jun 10;251(11):3480–3488. [PubMed] [Google Scholar]
  17. Stahl D. A., Walker T. A., Meyhack B., Pace N. R. Precursor-specific nucleotide sequences can govern RNA folding. Cell. 1979 Dec;18(4):1133–1143. doi: 10.1016/0092-8674(79)90226-5. [DOI] [PubMed] [Google Scholar]
  18. Walker G. C., Uhlenbeck O. C., Bedows E., Gumport R. I. T4-induced RNA ligase joins single-stranded oligoribonucleotides. Proc Natl Acad Sci U S A. 1975 Jan;72(1):122–126. doi: 10.1073/pnas.72.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES