An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron

Nucleic Acids Res. 1996 Apr 1;24(7):1337-44. doi: 10.1093/nar/24.7.1337.

Abstract

On the basis of the nucleotide sequence of Tetrahymena group I intron, we constructed a 31 residue RNA that has the P7 stem and the 3'-terminal guanosine residue (3'-G) with a putative stem-loop structure (P9.0) intervening between them. For this model RNA (P7/P9.0/G), four residues around the guanosine binding site (GBS) in the P7 stem were found to exhibit much lower sensitivities to ribonuclease V1 than those of a variant RNA having adenosine in place of the 3'-G, suggesting that the 3'-G contacts around the GBS. NMR analyses of the imino proton resonances of the P7/P9.0/G RNA indicated that the base pairing in the GBS is retained on the interaction with the 3'-G, and that the two base pairs of the putative P9.0 stem-loop are definitely formed. Comparison of the RNA with its variants with either A (3'-A) or a deletion in place of the 3'-G suggested that the stability of the P9.0 stem-loop is affected by the GBS-3'-G interaction. The melting temperatures of the P9.0 stem-loop were determined from the UV absorbances of these RNAs, which quantitatively indicated that the P9.0 stem-loop is significantly stabilized by the interaction of the GBS with the 3'-G, rather than the 3'-A, and also by direct interaction with divalent cations (Mg2+, Ca2+ or Mn2+). Upon replacement of the G-C base pair by C-G in the GBS of the P7/P9.0/G RNA, the specificity was switched from 3'-G to 3'-A, as in the case of the intact intron.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cations, Divalent / chemistry
  • Hydrogen Bonding
  • Introns*
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • RNA Splicing*
  • RNA, Protozoan / chemistry
  • RNA, Protozoan / genetics*
  • Tetrahymena / genetics*

Substances

  • Cations, Divalent
  • RNA, Protozoan