Conformational Dynamics of Single pre-mRNA Molecules During \u3cem\u3eIn Vitro\u3c/em\u3e Splicing

The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro, we observed a multitude of generally reversible time-and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium

Broad-spectrum aptamer inhibitors of HIV reverse transcriptase closely mimic natural substrates

A detailed understanding of how aptamers recognize biological binding partners is of considerable importance in the development of oligonucleotide therapeutics. For antiviral nucleic acid aptamers, current models predict a correlation between broad-spectrum inhibition of viral proteins and suppression of emerging viral resistance, but there is little understanding of how aptamer structures contribute to recognition specificity. We previously established that two independent single-stranded DNA aptamers, R1T and RT1t49(−5), are potent inhibitors of reverse transcriptases (RTs) from diverse branches of the primate lentiviral family, including HIV-1, HIV-2 and SIV(cpz). In contrast, class 1 RNA pseudoknots, such as aptamer T1.1, are specific for RTs from only a few viral clades. Here, we map the binding interfaces of complexes formed between RT and aptamers R1T, RT1t49(−5) and T1.1, using mass spectrometry-based protein footprinting of RT and hydroxyl radical footprinting of the aptamers. These complementary methods reveal that the broad-spectrum aptamers make contacts throughout the primer-template binding cleft of RT. The double-stranded stems of these aptamers closely mimic natural substrates near the RNase H domain, while their binding within the polymerase domain significantly differs from RT substrates. These results inform our perspective on how sustained, broad-spectrum inhibition of RT can be achieved by aptamers

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

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.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62205/1/mditzler_1.pd

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

Despite numerous structural and biochemical 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 a total of ∼300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected “gain of function” mutation, which is validated by native gel electrophoretic mobility shift assay. We observe that active site nucleobases and water molecules stabilize a geometry favorable to catalysis through a dynamic hydrogen bonding network. Simulations in which A38 is unprotonated show its N1 move into close proximity of the active site 2′-OH, indicating that A38 may act as a general base during cleavage, a role that has generally been discounted due to the longer distances observed in crystal structures involving inactivating substrate analogs. By contrast, simulations in which N1 of A38 is protonated place N1 in close proximity to the 5′-oxygen leaving group, which supports the proposal that A38 serves as a general acid. In analogy to protein enzymes, we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 2′-OH to the 5′-oxygen. Furthermore, our simulations suggest an important role for protonation of N1 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 long residency water molecules in transition-state stabilization

Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, “UCAA-motif” RNA Aptamers

RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs. The screen also identified a new, nonpseudoknot motif with a conserved unpaired UCAA element. High-throughput sequence (HTS) analysis identified 181 clusters capable of forming this novel element. Comparative sequence analysis, enzymatic probing and RT inhibition by aptamer variants established the essential requirements of the motif, which include two conserved base pairs (AC/GU) on the 5′ side of the unpaired UCAA. Aptamers in this family inhibit RT in primer extension assays with IC50 values in the low nmol/l range, and they suppress viral replication with a potency that is comparable with that of previously studied aptamers. All three known anti-RT aptamer families (pseudoknots, the UCAA element, and the recently described “(6/5)AL” motif) are therefore suitable for developing aptamer-based antiviral gene therapies