P-RNase

The ribozymes were widely modified and can be further subdivided according to their structural features in group I ribozymes, hammerhaed ribozymes, hairpin ribozymes, ribonucelase P (RNase P), and hepatitis delta virus ribozymes. 

Carefully discard the supernatant, add 200 pi of 75% ethanol, and centrifuge at top speed for 20 min at 4°. Discard supernatant and resuspend in 150 p RNase-free water. 

Poly-C-SpecificRibonuclease (P-RNase) (EC3.1.27.5). Warshawand Fournier (W3) showed that an increase in plasma enzyme activity of pancreatic P-RNase in patients with AP may indicate necrotic lesions, and is one of the few direct markers of pancreatic tissue injury (Nl, W4). Due to the time-consuming and cumbersome nature of the P-RNase assay procedure and the development of effective visualization techniques providing direct information on the structure of the inflamed pancreas, the diagnostic utility of the P-RNase assay has not been extensively studied (Table 3). 

RNase P. RNase P is a trans-acting ribozyme (150-500 nucleotides) that processes pre-tRNAs by endonucleolytically cleaving off leader sequences from the 5 -end. RNase P positions and activates a water molecule to attack the scissile phosphate. 

P (RNAse P), which catalyzes the maturation of tRNA by removing nucleotides from the 5 end of the precursor molecule (Section 28.1.8). Finally, as we shall see in Chapter 29. the RNA component of ribosomes is the catalyst that carries out protein synthesis. 

Ribonuclease P (RNase P) consists of both protein and an RNA component that has catalytic activity. RNase P functions in eukaryotic cells to process the 5 end of precursor tRNA molecules. RNase P also can be directed to cleave any RNA molecule when the target is complexed with a short complementary oligonucleotide called an external guide. 

A 5 sequence of variable length that Is absent from mature tRNAs is present in all pre-tRNAs (Figure 12-36). These extra 5 nucleotides are removed by ribonuclease P (RNase P), a ribonucleoprotein endonuclease. Studies with E. coll RNase P indicate that at high Mg concentrations, the RNA component alone can recognize and cleave E. coll pre-tRNAs. The RNase P polypeptide increases the rate of cleavage by the RNA, allowing it to proceed at physiological Mg concentrations. A comparable RNase P functions In eukaryotes. 

Some nucleic acids are capable of self-splicing. These catalytic DNA and RNA are known as deoxyribozymes (Li and Breaker, 1999 Sheppard et al, 2000) and ribozymes (Doherty and Doudna, 2000 Scott and Klug, 1996) respectively. The 3 - and/or 2 -hydroxyls of DNA/RNA serve as a catalytic site that invariably requires a metal ion for the catalytic activity. Deoxyribozymes are quasi-catalytic while ribozymes can be catalytic, e.g. ribonuclease P (RNase P) as well as quasi-catalytic, e.g. introns and hammerhead RNAs. RNase P resources are maintained at http //www.mbio.ncsu.edu/RnaseP/homeJitml... 

Ribonudease P RNA. Ribonuclease P (RNase P) is the endoribonuclease that generates the mature 5 -ends of tRNA by removal of the 5 -leader elements of pre-tRNA. Haloenzymes are composed of essential RNA and protein subunits. E. coli RNase P contains a protein subunit of 119 amino acids and a single RNA molecule of 377 nucleotides, with RNA exhibiting multiple turnovers in vitro and in vivo, to process tRNA precursors with extended 5 terminal sequences (Frank and Pace, 1998 Xiao et al, 2002). Although the RNA subunit of cellular RNase P contains the catalytic active site (catalyzing maturation of tRNA in vitro), the protein subunit is required for in vivo activity. 

The RNA mini-helix, consisting solely of T and acceptor stem (and including a 5 leader), is cleaved by RNase P. RNase P occupies a patch on the substrate that overlaps the acceptor and T stems. 

Prokaryotic pre-rRNA processing The E. coli primary rRNA transcript contains 16S rRNA, 1 or 2tRNA, 23S rRNA and 5S rRNA and some with 1 or 2 more tRNA (in order from 5 to 3 ends). The trimming and processing of pre-rRNA to mature rRNA involves various RNases such as RNase HI, RNase P, RNase E, RNase F and RNase M at different stages. 

Brown P.H., Bruckenstein D.A., Han-VEY J.C., et al.An assessment of the anti-sense properties of RNase FI-competent and steric-blocking oligomers. Nucleic Acids Res. 1995 23 1197-1203. 

The association between RNase A and 3 -UMP or 3 -dUMP has been studied by P n.m.r. and kinetic methods, respectively. In both cases the participation of two dissociable groups at the active site of the enzyme was demonstrated, in agreement with n.m.r. and A -ray - studies on the binding of 3 -CMP to RNase. In the binding of Tj RNase to purine nucleotide monophosphates, the phosphate group appears to have an important effect while the ribose ring is relatively unimportant. ... 

The crystal structure of one LRR protein, the RNAse inhibitor, has revealed that leucine-rich repeats correspond to p-a structural units. This units are arranged for a parallel p-sheet with one surface exposed to solvent so that the protein acquires an unusual non-globular shape, which may be responsible for proteinbinding functions [57]. 

The effect of formalin-treatment on the structural properties of RNase A was examined using circular dichroism (CD) spectropolarimetry. A brief introduction to CD spectropolarimetry is provided in Section 15.15.2 for those readers unfamiliar with this biophysical method. The secondary structure of RNase A consists of one long four-stranded anti-parallel p-sheet and three short a-helixes,44 which places RNase A in the a + p structural class of proteins. The effect of a 9-day incubation of RNase A (6.5mg/mL) in 10% formalin on the protein secondary structure was examined with CD spectropolarimetry in the far-UV region (170-240nm) as shown in Figure 15.6a. The resulting... 

These spectra are similar to that of native RNase A at 95°C (not shown). The far-UV spectrum at 95°C indicates a retention of substantial p-sheet secondary structure, but a significant loss of the a-helix conformation as indicated by the decrease of intensity at 222 nm.48 The near-UV spectrum at 95°C indicates a complete collapse of tertiary structure as is seen in molten globule proteins.49 Trace 3 is the sample from trace 2 after cooling the protein to 23°C. Both spectra reveal little recovery of either secondary or tertiary protein structure. 

Monoparametric DNA Analysis Fixed cells were stained with PI 25 pg/niL, RNase 5 pg/mL, Nonidet P 40 0.125 pg/mL. Cells were kept at RT for 60 in the dark and were analyzed by BD FACSCalibur flow cytometer. Aggregates were gating out by DDM papameters and DNA content analysis was performed in >10,000-gated cells. DNA histograms were analyzed using ModFit LT . 

Figure 5. (Continued) different panels (e and f for HeLa, j and k for chicken erythrocyte, and o and p for yeast). A section profile obtained along X-Y line shows a typical granular structure in die nucleus (e, j, o), and the peak-to-peak distance between the granular structure was distributed from 60 nm to 120 nm (e). The diickness of the chromatin fibers released out of die nucleus varied possibly due to the assembly of diinner fibers (f, k, p). A section profile for the spread fibers was obtained along X-Y line (f, k, p). Isolated HeLa cell nucleus was treated widi (r, s) or without (q) RNase. The treatment releases SOnmfiber from the nucleus. The histogram of die fiber width is shown in an inset of (s). Bars, 250 nm. (See Colour Plate 2.)...

The specific incorporation of selenium into nucleic acids was first identified by K. P. McConnell in the early 1970s. This initial identification was based on chromatographic separation of nucleobases and sensitivity to RNases. Soon after studies to determine the specificity of selenium incorporation into tRNA was carried out in... 

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