Folding and Conformational Dynamics of the Hairpin Ribozyme and the Spliceosome:Combining Computational and Experimental Analyses.

[摘要] The vital role of RNA structure and dynamics in determining biological function is increasingly appreciated throughout the life sciences.RNA-coding genes are now recognized to be far more abundant in eukaryotes than their protein-coding counterparts and are essential to the central biochemical processes within all living cells.Here, we use computational and experimental techniques in order to understand the folding and conformational dynamics of two vastly different RNA systems (the hairpin ribozyme and the spliceosome) at the single molecule level. Large energy barriers separating misfolded and functional states are a well appreciated characteristic of RNA. By contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction free of deeply dividing energy barriers.Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing, and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the folding energy landscape.Despite numerous investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed multiple molecular dynamics (MD) simulations. Our simulations suggest an important role for protonation of A38 in promoting a favorable geometry similar to that observed in transition-state analog crystal structures, and support previously proposed roles of A38, G8, and water in catalysis. Finally we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 2’-OH to the 5’-oxygen. Despite over 20 years of study, the kinetic and structural details of spliceosome assembly are not well understood.To track in real-time the conformational states through which the spliceosome takes a pre-mRNA, we have developed an smFRET based spliceosome assembly and activity assay. Our data quantify the kinetics of ATP, small nuclear RNA, and sequence dependent pre-mRNA conformational changes.We find that wild-type pre-mRNA inhabits a broad and dynamic conformational space and is specifically funneled into a constrained conformational sub-space by ATP-dependent, spliceosome induced processes.

[发布机构] University of Michigan

[效力级别] Spliceosome

[关键词] Ribozyme;Spliceosome;Single Molecule;FRET;Molecular Dynamics;Biological Chemistry;Science;Biophysics