Group II introns are mu
ltidomain ribozymes that cata
lyze their own remova
l from pre-mRNA. The nuc
leophi
le for the first c
leavage step is the 2'OH of a specific adenosine within domain 6 (D6), ca
lled the branch site. Mechanistic para
lle
ls and
limited secondary structura
l simi
larity with the eukaryotic sp
liceosome
lead many to specu
late that the two systems have a common ancestry. We have e
lucidated structura
l features of the branch site region and the importance of the interna
l loop to branch site conformation within D6 of the ai5
Group II intron by NMR and f
luorescence spectroscopy. F
luorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to so
lvent and that this position is enhanced by Mg
2+ or Ca
2+. Upfie
ld NMR chemica
l shifts of imino protons of the two uridine residues f
lanking the branch site adenosine, and an
n n + 2 NOE between them, suggest a stacked intrahe
lica
l conformation of the two uridines. In contrast, resu
lts of NMR and 2-aminopurine f
luorescence spectra of a mutated D6 from which the interna
l loop had been de
leted suggest a
less exposed position of the branch site adenosine, which is
like
ly to form a G-A base pair with the opposing 3'G. These findings describe a mode
l in which the branch site adenosine of D6 is in an extrahe
lica
l position, surrounded by two intrahe
lica
l bases. The interna
l loop and diva
lent meta
l ions faci
litate this motif.