Bacterial RNase P proteins and their use in identifying antibacterial compounds

• US 6,936,432 B2
• Filed: 03/01/2001
• Issued: 08/30/2005
• Est. Priority Date: 03/01/2000
• Status: Expired due to Fees

• Current Assignee: Ohio State University
• Original Assignee: Message Pharmaceutical Incorporated
• Inventors: Jovanovic, Milan, Gopalan, Venkat, Xavier, K. Asish, Eder, Paul S., Giordano, Tony, Powers, Gordon D.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US09/798,635
• Publication Number:
  US 20040127480A1
• Time in Patent Office: 1,643 Days
• Field of Search: 435/199, 435/18
• US Class Current: 435/18
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention features novel RNase P molecules and nucleic acids encoding the same. Methods for discovery of antimicrobial compounds are also featured.

Claims

1. An isolated polypeptide comprising an RNase P consensus sequence, said polypeptide comprising the amino acid sequence of SEQ ID NO:

55, wherein said polypeptide has RNase P protein activity.

2. A method of identifying an antibiotic agent, said method comprising:

i) contacting an RNase P holoenzyme comprising the polypeptide of claim 1 with an RNase P substrate in the presence and in the absence of a compound; and

ii) measuring the enzymatic activity of said holoenzyme;

wherein a compound is identified as an antibiotic agent if said compound produces a detectable decrease in said RNase P enzymatic activity as compared to activity in the absence of said compound.

3. The method of claim 2, wherein said activity is measured by fluorescence spectroscopy.

4. The method of claim 3, wherein said RNase P substrate is fluorescently tagged ptRNA^Gln.

5. The method of claim 3, wherein said contacting is carried out in a buffer comprising 10-40 μ

g/ml carbonic anhydrase and 10-100 μ

g/ml polyC.

6. The method of claim 5, wherein said buffer further comprises at least one of the following:

0.5-5% glycerol;

10-100 μ

g/ml hen egg lysozyme;

10-50 μ

g/ml tRNA;

or

1-10 mM DTT.

7. The method of claim 2, wherein said polypeptide, when combined with an RNA subunit to form an RNase P holoenzyme, has at least 20% of the enzymatic activity of an E. coli or B. subtilis RNase P holoenzyme.

8. A method of identifying an antibiotic agent, said method comprising:

i) contacting an RNase P holoenzyme comprising an RNase P polypeptide and having RNase P protein activity with an RNase P substrate in the presence and in the absence of a compound, wherein said RNase P polypeptide comprises the sequence set forth in SEQ ID NO;

55; and

iii) measuring the enzymatic activity of said holoenzyme, said measuring comprising determining the fluorescence polarization level of a fluorescently tagged oligonucleotide that hybridizes to either the cleaved or intact form of said substrate;

wherein said compound is identified as an antibiotic agent if said compound produces a detectable decrease in said RNase P enzymatic activity as compared to activity in the absence of said compound.

9. The method of claim 8, wherein said RNase P substrate is ptRNA^Gln.

10. The method of claim 8, wherein said polypeptide, when combined with an RNA subunit to form an RNase P holoenzyme, has at least 20% of the enzymatic activity of an E. coli or B. subtilis RNase P holoenzyme.

11. A method of identifying a polypeptide with an RNase P polypeptide consensus sequence, said method comprising the steps of:

(a) identifying a polypeptide that has sequence identity to a known RNase P polypeptide; and

(b) determining if said identified polypeptide of step (a) conserves at least nine of the following twenty amino acids in the E. coli RNase P protein sequence;

R11, L12, F18, R46, G48, V51, K53, K54, A59, V60, R62, N63, K66, R67, R70, L80, D84, V86, L101, and L105, wherein the numbering of said twenty amino acids refers to that of E. coli RNase P, and wherein a polypeptide that does conserve at least nine of said twenty amino acids in the E. coli RNase P protein sequence is a polypeptide with an RNase P consensus sequence.

12. The method of claim 11, wherein said polypeptide comprises the following twenty amino acids:

R11, L12, F18, R46, G48, V51, K53, K54, A59, V60, R62, N63, K66, R67, R70, L80, D84, V86, L101, and L105, wherein the numbering refers to the position of the amino acid in the sequence of E. coli RNase P.

13. The method of claim 11, wherein said identified polypeptide, when combined with an RNA subunit to form an RNase P holoenzyme, has at least 20% of the enzymatic activity of an E. coli or B. subtilis RNase P holoenzyme.

14. The method of claim 11, wherein said identified polypeptide is a bacterial polypeptide.

15. The method of claim 14, wherein said identified polypeptide is not a polypeptide from one of the following organisms:

Coxiella burnetii (None Mile) U10529, Rickettsia prowazekii (Madrid E) AJ235272, Neisseria meningitidis (Z2491) AL162753, Neisseria meningitidis (MC58) AE002540, Buchnera aphidocola M80817, Buchnera aphidocola (SGS) AF008210, Buchnera sp. (APS) AP000398, Haemophilus influenza (RD KW20) U32848, Escherichia coli M11056, Escherichia coli (K-12) AE000394, Proteus mirabilis M58352, Pseudomonas aeruginosa (PAO1) AE004968, Pseudomonas putida P25752, Salmonella typhi (CT18), Yersinia pestis (Orientalis), Xyelella fastidiosa AE004083, Campylobacter jejuni (NCTC

     11168) AL139076, Helicobacter pylori (26695) AE000645, Helicobacter pylori (J99) AE001557, Micrococcus luteus (S66) U64884, Mycobacterium avium (104) AF222789, Mycobacterium bovis (AF2122/97), Mycobacterium leprae (Lortist

     6) L39923, Mycobacterium tuberculosis (H37Rv) AL021426 X92504, Streptomyces bikiniensis (Zorbonenis) M83112, Streptomyces coelicolor (A3(2)) M82836 AL049826 AF031590, Bacillus halodurans (C-125) AB013492, Bacillus subtilis (168) X62539 AL009126, Mycoplasma capricolum (mcs5) P14982, Mycoplasma genitalium (G-37) U39713, Mycoplasma pneumoniae (M-129) U00089, Staphylococcus aureus (ISP3) AF135268, Ureaplasma urealyticum (3/1) AE002158, Pseudanabaena sp. (PCC6903) AJ000513 Synechocystis sp. (PCC6803) X81989, Borrellia burgdorferi (212) Z12166, Borrellia burgdorferi (B31) AE000783, Treponema pallidum (Nichols) P50069, Chlamydia trachomatis (serovar D) AE001351, Chlamydia muridarum (trachomatis MoPn) AE002160, Chlamydophila pneumoniae (CWL

     029) AE001673, Chlamydophila pneumoniae (AR39) AE002251, Deinococcus radiodurans (R1) AE002049, Thermotoga maritima (MSB8) AAD36531, B. burgdorferi, B. burgdorferi-partial, C. burnetii, C. pneumoniae-2, C. trachomatis, H. influenza, H. pylori-48, M. leprae, M. luteus, M. tuberculosis-2, M. bovis, Pseudanabaena-6903, R. prowazeki, S. bikiniensis, Synechocystis 6803, Staphylococcus aureus, and S. pneumoniae.