14 research outputs found
Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester
We analyzed cleavage of precursor tRNAs with an LNA, 2ā²-OCH3, 2ā²-H or 2ā²-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2ā²-substituent at nt ā1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3ā² -endo ribose and without the H-bond donor function of the 2ā²-substituent. LNA and 2ā²-OCH3 suppressed processing at the major aberrant mā1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2ā²-OCH3 supports a model where the extra methylene (LNA) or methyl group (2ā²-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects
100The effect of unilateral obstruction and antiangiotensin II treatment on renal tubule and interstitial cell apoptosis and fibrosis in rats
Development of an Effective Method for Recovery of Viral Genomic RNA from Environmental Silty Sediments for Quantitative Molecular Detection āæ
Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol-chloroform-isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest rate of recovery (geometric mean of 11%, with a geometric standard deviation of 0.02; n = 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest rate of PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River, Miyagi, Japan, and its estuary from November 2007 to April 2009, and the genomic RNAs of enterovirus and human norovirus in these samples were quantified by reverse transcription (RT)-quantitative PCR (qPCR). The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 Ć 102 and 2.4 Ć 102 copies/g (wet weight), respectively. This is the first report to obtain quantitative data for a human pathogenic virus in a river and in estuarine sediments using RT-qPCR
NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P
Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5ā² end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn2+ bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08Ā ā«. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn2+ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new āmetal cocktailā chemical shift perturbation strategy involving titrations with , Zn2+, and confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution