An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron.
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.
Watanabe
S
S
Department of Biophysics and Biochemistry, School of Science, University of Tokyo, Japan.
Kawai
G
G
Muto
Y
Y
Watanabe
K
K
Inoue
T
T
Yokoyama
S
S
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D006860:Hydrogen Bonding; D007438:Introns; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D013768:Tetrahymena / Q000235:genetics*
D002413:Cations, Divalent; D016053:RNA, Protozoan