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US 6,649,744 B1Granted
Rnase P polypeptides, polynucleotides, and methods using their mechanisms of action
AssigneeSmithKline Beecham Corporation
InventorsMichael J. Gress, Lisa A Hegg, Hu Li, Joseph J. Park
Life of this patent
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Abstract
This invention relates to a novel bacterial ribonucleoprotein complex and the component parts thereof. More specifically, this invention relates to RNase P RNA isolated from Staphylococcus aureus and the use of RNase P RNA in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy.
Claims2
An antagonist that inhibits or an agonist that activates an activity of an RNA selected from the group consisting of:
- an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of:
- Staphylococcus aureus RNaseP Km=53±4 nM determined in a reaction in 1×buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate;
- Staphylococcus aureus RNaseP kcat=3.4±0.1 min−1 determined in a reaction in 1×buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate;
- Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol;
- Staphylococcus aureus RNaseP V1 cleavage of nucleotides selected from the group consisting of:
- 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294;
- and
- Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of:
- 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177;
- and
- said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of:
- 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271 (bulged A).
9 more elements
An antagonist that inhibits an activity of a polypeptide selected from the group consisting of:
- an RNA transcribed from a polynucleotide comprising an nucleotide sequence having at least a 90% identity to the amino acid sequence of SEQ ID NO:3, and an RNA transcribed from a polynucleotide comprising an nucleotide sequence set forth in SEQ ID NO:3, wherein said activity is selected from the group consisting of:
- Staphylococcus aureus RNaseP Km=53±4 nM determined in a reaction in 1×buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe a substrate;
- Staphylococcus aureus RNaseP kcat=3.4±0.1 min−1 determined in a reaction in 1×buffer comprising 100 mM Tris-Cl, pH7.0, 150 mM KCl, 10 mM MgCl2, 5% PEG, and [E]=20 nM using cloned pre-tRNAphe as a substrate;
- Staphylococcus aureus RNaseP RNA binding isotherm of Kd=8±1 nM for RNaseP protein determined in a reaction buffer comprising 20 mM K-Hepes, pH8.0, 0.01% NP-40, 400 mM NH4OAc, 10 mM MgCl2, and 5% glycererol;
- Staphylococcus aureus RNaseP V 1 cleavage of nucleotides selected from the group consisting of:
- 8-11, 16-23, 30-33, 37-54, 64-84, 96-105, 119-130, 150-156, 166-169, 181-184, and 290-294;
- and
- Staphylococcus aureus RNase T2 cleavage of nucleotides selected from the group consisting of:
- 24-27, 55-60, 86-91, 106-108, 135-138, and 170-177;
- and said compound modulates the DEPC cleavage of nucleotides selected from the group consisting of:
- 159-161, A211-213.A203, 204, and A169, 292-294 (L15.2), 271(bulged A).
8 more elements
Dependencies are inferred from the claim text.