Hydrostatic and osmotic pressure study of the RNA hydration

The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNAPhe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNAPhe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86

Comparative genomics of metabolic capacities of regulons controlled by cis-regulatory RNA motifs in bacteria

BACKGROUND: In silico comparative genomics approaches have been efficiently used for functional prediction and reconstruction of metabolic and regulatory networks. Riboswitches are metabolite-sensing structures often found in bacterial mRNA leaders controlling gene expression on transcriptional or translational levels. An increasing number of riboswitches and other cis-regulatory RNAs have been recently classified into numerous RNA families in the Rfam database. High conservation of these RNA motifs provides a unique advantage for their genomic identification and comparative analysis. RESULTS: A comparative genomics approach implemented in the RegPredict tool was used for reconstruction and functional annotation of regulons controlled by RNAs from 43 Rfam families in diverse taxonomic groups of Bacteria. The inferred regulons include ~5200 cis-regulatory RNAs and more than 12000 target genes in 255 microbial genomes. All predicted RNA-regulated genes were classified into specific and overall functional categories. Analysis of taxonomic distribution of these categories allowed us to establish major functional preferences for each analyzed cis-regulatory RNA motif family. Overall, most RNA motif regulons showed predictable functional content in accordance with their experimentally established effector ligands. Our results suggest that some RNA motifs (including thiamin pyrophosphate and cobalamin riboswitches that control the cofactor metabolism) are widespread and likely originated from the last common ancestor of all bacteria. However, many more analyzed RNA motifs are restricted to a narrow taxonomic group of bacteria and likely represent more recent evolutionary innovations. CONCLUSIONS: The reconstructed regulatory networks for major known RNA motifs substantially expand the existing knowledge of transcriptional regulation in bacteria. The inferred regulons can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. The obtained genome-wide collection of reference RNA motif regulons is available in the RegPrecise database (http://regprecise.lbl.gov/)

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity – a “pinch” that results in rate acceleration by almost three orders of magnitude. Kinetic analysis of ribozymes with systematically altered length and stability of the peripheral domain revealed that about one third of its free energy of formation is used to lower an activation energy barrier, likely related to a rate-limiting conformational change leading to the pre-catalytic state. These findings provide a quantitative view of enzyme regulation by peripheral domains and may shed light on the energetics of allosteric regulation