Conformation of the Group II intron branch site in solution.
Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2'OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5gamma Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+ or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and an n --> n + 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G-A base pair with the opposing 3'G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.
Department of Chemistry and Biochemistry, Florida State University, 214 DLC, Tallahassee, Florida 32306-4390, USA.
Jörg C
JC
Schlatterer
Samuel H
SH
Crayton
Nancy L
NL
Greenbaum
D015075:2-Aminopurine / Q000737:chemistry; D000241:Adenosine / Q000737:chemistry; D001483:Base Sequence; D007438:Introns / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D044563:RNA, Ribosomal, Self-Splicing / Q000737:chemistry* / Q000235:genetics; D012996:Solutions
D044563:RNA, Ribosomal, Self-Splicing; D012996:Solutions; D015075:2-Aminopurine; D000241:Adenosine