Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including =10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNAAsp by a factor of 10(4) as determined from both the ratio of the pre-tRNAAsp dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNAAsp. Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing. Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.
Aptamers (protein binding oligonucleotides) have potential as a new class of targeted therapeutics. For applications requiring chronic systemic administration, aptamers must achieve high-affinity target binding while simultaneously retaining high in vivo stability, tolerability, and ease of chemical synthesis. To this end, we describe a method for generating aptamers composed entirely of 2'-O-methyl nucleotides (mRmY). We present conditions under which 2'-O-methyl transcripts can be generated directly and use these conditions to select a fully 2'-O-methyl aptamer from a library of 3 x 10(15) unique 2'-O-methyl transcripts. This aptamer, ARC245, is 23 nucleotides in length, binds to vascular endothelial growth factor (VEGF) with a Kd of 2 nM, and inhibits VEGF activity in cellular assays. Notably, ARC245 is so stable that degradation cannot be detected after 96 hr in plasma at 37 degrees C or after autoclaving at 125 degrees C. We believe ARC245 has considerable potential as an antiangiogenesis therapeutic.
Ribonuclease P (RNase P) is the endoribonuclease responsible for the 5'-maturation of precursor tRNA transcripts. In bacteria, RNase P is composed of a catalytic RNA subunit and an associated protein subunit that enhances the substrate specificity of the holoenzyme. We have initiated a study of the biophysical properties of the protein subunit from Bacillus subtilis RNase P (P protein) toward the goal of understanding the thermodynamics of RNase P holoenzyme assembly. The P protein is predominantly unfolded in 10 mM sodium cacodylate at neutral pH based on circular dichroism and NMR studies and therefore has several characteristics typical of "intrinsically unstructured" proteins. Furthermore, the P protein folds to its native alpha/beta structure upon addition of various small molecule anions. Anion-induced folding is best attributed to the binding of these anions to the folded state of the protein, and a model is presented which describes the observed tightly coupled folding and binding phenomena. The P protein also undergoes a cooperative folding transition upon addition of the osmolyte trimethylamine N-oxide (TMAO). The equilibrium constant of folding (K(fold)) at 37 degrees C for the P protein was determined to be 0.0071 +/- 0.0005 using a two-state folding model to describe the TMAO titration data. Thus, the folding and binding equilibria observed in the anion-induced folding of the P protein can be uncoupled to determine the intrinsic binding affinities (K(a)'s) of the anionic ligands. Evidence that the osmolyte-induced and the ligand-induced folded conformations of the P protein are structurally similar is also presented.
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