Molecular recognition properties of IGS-mediated reactions catalyzed by a Pneumocystis carinii group I intron.
We report the development, analysis and use of a new combinatorial approach to analyze the substrate sequence dependence of the suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS)-mediated reactions is not high. In addition, the sequence specificity of suicide inhibition decreases with increasing MgCl(2) concentration, reverse cyclization is substantially more sequence specific than suicide inhibition, and multiple reverse cyclization products occur, in part due to the formation of multiple cyclization intermediates. Thermodynamic analysis reveals that a base pair at position -4 of the resultant 5' exon-IGS (P1) helix is crucial for tertiary docking of the P1 helix into the catalytic core of the ribozyme in the suicide inhibition reaction. In contrast to results reported with a Tetrahymena ribozyme, altering the sequence of the IGS of the P.carinii ribozyme can result in a marked reduction in tertiary stability of docking the resultant P1 helix into the catalytic core of the ribozyme. Finally, results indicate that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.
Johnson
Ashley K
AK
Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Baum
Dana A
DA
Tye
Jesse
J
Bell
Michael A
MA
Testa
Stephen M
SM
D001483:Base Sequence; D001667:Binding, Competitive; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011010:Pneumocystis / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D004271:DNA, Fungal; D009841:Oligonucleotides; D016337:RNA, Catalytic; D015636:Magnesium Chloride