More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.
Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.
Chu
V T
VT
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Liu
Q
Q
Podar
M
M
Perlman
P S
PS
Pyle
A M
AM
D000241:Adenosine / Q000235:genetics; D001483:Base Sequence; D004722:Endoribonucleases; D007438:Introns; D007700:Kinetics; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D017423:Sequence Analysis, RNA; D017384:Sequence Deletion
D012313:RNA; D004722:Endoribonucleases; D000241:Adenosine