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RNA Enzymes: From Folding to Function in Living Cells

2008-03-25 , Fedor, Martha J.

Our research aims to generate fundamental insights into catalysis by RNA enzymes and into the pathways through which RNAs form specific functional structures. RNA catalysis remains an intriguing puzzle that has grown in significance since the recent discoveries that the ribosome itself is an RNA enzyme and that human and bacterial mRNAs contain self-cleaving ribozymes. The hairpin ribozyme catalyzes a reversible self-cleavage reaction in which nucleophilic attack of a ribose 2’ hydroxyl on an adjacent phosphorus proceeds through a trigonal bipyramidal trasition state that leads to the formation of 2’,3’-cyclic phosphate and 2’ hydroxyl termini. The metal cation independence of activity and the availability of high-resolution active site structures have made the hairpin ribozyme the prototype for nucleobase-mediated catalytic chemistry. A network of stacking and hydrogen bonding interactions align the reactive phosphate in the appropriate orientation for an SN2-type nucleophilic attack and orient nucleotide base functional groups near the reactive phosphate to facilitate catalytic chemistry. Two active site nucleobases, G8 and A38, adopt orientations reminiscent of the histidine residues that mediate general acid base catalysis in ribonuclease A, a protein enzyme that catalyzes the same phosphodiester cleavage chemistry. However, our biochemical experiments argue against analogous roles for G8 and A38 in hairpin ribozyme catalysis and suggest that these residues contribute to catalysis through positioning and orientation and electrostatic stabilization of the electronegative transition site. Ribozymes are useful model systems for investigation of RNA folding, since self-cleavage reflects the assembly of a precise functional structure. To learn how structure-function principles revealed through in vitro experiments translate to the behavior of RNA in living cells, we devised a way to evaluate RNA assembly in vivo using RNA self-cleavage rates to quantify assembly of functional RNA structures. Results of these studies show that intracellular RNA folding kinetics and equilibria are indistinguishable from RNA folding behavior in vitro, provided that in vitro folding reactions approximate the ionic conditions characteristic of an intracellular environment. These studies contribute basic knowledge of RNA structure and function and provide a framework for developing technical and therapeutic application involving RNAs as targets and reagents.

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Systems Biology and its Role in Predictive Health and Personalized Medicine

2008-02-05 , Voit, Eberhard O.

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Refinement of reduced protein models with all-atom force fields - Ph.D. Defense

2007-10-30 , Wróblewska, Liliana

The goal of the following thesis research was to develop a systematic approach for the refinement of low-resolution protein models, as a part of the protein structure prediction procedure. Significant progress has been made in the field of protein structure prediction and the contemporary methods are able to assemble correct topology for a large fraction of protein domains. But such approximate models are often not detailed enough for some important applications, including studies of reaction mechanisms, functional annotation, drug design or virtual ligand screening. The development of a method that could bring those structures closer to the native is then of great importance. The minimal requirements for a potential that can refine protein structures is the existence of a correlation between the energy with native similarity and the scoring of the native structure as being lowest in energy. Extensive tests of the contemporary all-atom physics-based force fields were conducted to assess their applicability for refinement. The tests revealed flatness of such potentials and enabled the identification of the key problems in the current approaches. Guided by these results, the optimization of the AMBER (ff03) force field was performed that aimed at creating a funnel shape of the potential, with the native structure at the global minimum. Such shape should facilitate the conformational search during refinement and drive it towards the native conformation. Adjusting the relative weights of particular energy components, and adding an explicit hydrogen bond potential significantly improved the average correlation coefficient of the energy with native similarity (from 0.25 for the original ff03 potential to 0.65 for the optimized force field). The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. The new, optimized potential was subsequently used to refine protein models of various native-similarity. The test employed 47 proteins and 100 decoy structures per protein. When the lowest energy structure from each trajectory was compared with the starting decoy, we observed structural improvement for 70% of the models on average. Such an unprecedented result of a systematic refinement is extremely promising in the context of high-resolution structure prediction.

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Positive Selection on Non-coding Sequences During Human Evolution: From Genome to Nucleotide

2008-03-04 , Wray, Gregory

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Mechanisms of Chromosomal Fragility and Rearrangements Triggered by Human Unstable Repeats

2008-01-22 , Lobachev, Kirill

Research of my lab focuses on understanding how chromosomal rearrangements arise and lead to hereditary diseases and cancer. Chromosomes containing repeats that can adopt stable secondary structures are highly prone for double-strand breaks and various types of rearrangements. Molecular mechanisms for this type of genetic instability in eukaryotes are poorly understood. Using yeast, S. cerevisiae, we are investigating the chromosomal fragility mediated by two sequence motifs: cruciform-forming inverted repeats and H-DNA-forming GAA/TTC triplet repeats. Both types of repeats strongly induce breakage which results from the replication arrest by the secondary structures. However, genetic requirements for fragility, mode of breakage and consequences for the genome integrity are different for these two types of repeats. We propose that the nature of the secondary structure predisposes chromosomes for the specific pattern of gross chromosomal rearrangements. These rearrangements are strikingly similar to carcinogenic aberration suggesting that repeat-mediated instability might be a general phenomenon that operates not only in yeast but also in humans. I will present recent data from my lab on proteins that are involved into the fragility.

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Better living through biosensors

2008-02-19 , Plaxco, Kevin

The ideal sensor will be sensitive, specific, versatile, small enough to hold in your hand, and selective enough to work even when faced with complex, contaminant-ridden samples. Given the affinity, specificity and generalizability of biomolecular recognition, biosensors have been widely touted for their potential to meet these challenging goals. To date, however, the translation of protein- and DNA-binding events into convenient, highly selective sensing platforms has proven problematic. We have solved this problem by employing the ligand-induced folding of proteins, peptides and DNA as a robust signal transduction mechanism. Our folding-based sensors are rapid (minutes to seconds), sensitive (micromolar to femtomolar), fully electronic, and generalizable to an enormous range of protein, nucleic acid and small molecule targets. The sensors are also reagentless, greater than 99% reusable, and selective enough to be employed in (and re-used from) blood, soil and other grossly contaminated materials. Because of their sensitivity, background suppression, operational convenience and impressive scalability folding-based biosensors appear ideally suited for electronic, on-chip applications in pathogen detection, proteomics and genomics.

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Hybrid Experiments: Linking Real-Time Simulations to In Vitro Electrophysiology Experiments

2007-10-30 , Butera, Robert J.

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