Fluorescence-detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5' cleavage site into a high free energy binding mode in the Tetrahymena ribozyme.
Fluorescence-detected stopped flow kinetics are reported for binding of pyrene (pyr) labeled oligonucleotide substrates, pyrCUCUA and pyrCCUCUA, to the L-21 ScaI ribozyme from Tetrahymena thermophila. Both oligomer substrates contain a UA sequence that mimics the cleavage site where pG attacks the self-splicing group I intron from which the ribozyme was derived. Kinetics were measured in the presence and absence of saturating 5'-monophosphate guanosine substrate (pG) at 5 mM Mg2+ and 15 degrees C. In the absence of pG, binding of both oligonucleotide substrates is consistent with a one step mechanism involving only base pairing. Upon addition of pG, pyrCCUCUA is observed to bind in two steps: base pairing to the ribozyme to form the P1 helix and, presumably, subsequent docking of the P1 helix into the catalytic core. A third transient is also observed, which likely includes the chemical step following docking. All rate constants are measured for this mechanism. Surprisingly, the equilibrium constant for docking, K2, is unfavorable in the absence of pG (K2 < 1) and only modestly favorable in the presence of pG (K2 = 4). These results contrast with those for a 5' exon mimic, pyrCCUCU, in which docking is strongly favored under the above conditions in the absence of pG; K2 = 100 [Bevilacqua, P. C., Kierzek, R., Johnson, K. A., & Turner, D. H. (1992) Science 258, 1355-1358]. These results suggest an unfavorable interaction between the ribozyme and the pA at the site of cleavage. Implications are discussed for the catalytic strategy of the ribozyme and for the self-splicing cascade that occurs in nature.
Bevilacqua
P C
PC
Department of Chemistry, University of Rochester, New York 14627-0216, USA.
Li
Y
Y
Turner
D H
DH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011721:Pyrenes; D016337:RNA, Catalytic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000378:metabolism*
D009843:Oligoribonucleotides; D011721:Pyrenes; D016337:RNA, Catalytic