[show abstract][hide abstract] ABSTRACT: Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
Nucleic Acids Research 01/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3' end. Here we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3' end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.
PLoS ONE 01/2013; 8(1):e54384. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.
[show abstract][hide abstract] ABSTRACT: Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.
PLoS ONE 01/2012; 7(5):e38024. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA can be used as a structural component in the process of making conductive polymers called nanowires. Accurate molecular models could lead to a better understanding of how to prepare these types of materials. Here we present a computational tool that allows potential DNA-linked polymer designs to be screened and evaluated. The approach involves an iterative procedure that adjusts the positions of DNA-linked monomers in order to obtain reasonable molecular geometry compatible with normal DNA conformations and with the properties of the polymer being formed. This procedure has been used to evaluate designs already reported experimentally, as well as to suggest a new design based on pyrrylene vinylene (PV) monomers.
[show abstract][hide abstract] ABSTRACT: Fibrin polymerizes via noncovalent and dynamic association of thrombin-exposed "knobs" with complementary "holes." Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob "A" mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics.