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RNA interferases and methods of use thereof

A technology of endonuclease and activity, applied in the field of molecular biology

Inactive Publication Date: 2006-09-27
UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no evidence that Kid(PemK) inhibits the replication of chromosomal DNA

Method used

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  • RNA interferases and methods of use thereof
  • RNA interferases and methods of use thereof
  • RNA interferases and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0249] As described here, E. coli cells were permeabilized using toluene treatment and these cells were used to demonstrate that MazF inhibits translation, but not RNA synthesis or DNA replication. It was also shown that MazF cleaves mRNA specifically between the A and C residues of the ACA sequence in a ribosome-independent manner. Thus, the present invention demonstrates that MazF interferes with the function of mRNA by cleaving mRNA at a specific site. Therefore, the inventors of the present invention found that MazF is a novel endoribonuclease, and named it "mRNA interferase" herein.

[0250] Materials and methods

[0251] Strains and plasmids. Escherichia coli BL21(DE3), BW25113 (Datsenko and Wanner, Proc Natl Acad Sci USA 97, 6640-5 (2000)) and MRE600 (Swaney et al., Antimicrob Agents Chemother 42, 3251-5 (1998)) were used. Plasmid pET-21cc-MazEF was constructed from plasmid pET-21cc (Novagen), which was modified to express MazE and MazF(His) under the control of the ...

Embodiment II

[0288]Importantly, the cellular targets of MazF had not been identified prior to the discovery of the present invention. As shown here, MazF functions as a highly sequence-specific endoribonuclease that cleaves cellular mRNA at the ACA site. This activity may effect partial or total inhibition of protein synthesis in the cell. Based on standard calculations based on the same principle that the probability of incorporation of any of the four nucleotides at each of the three nucleotide positions in the ACA sequence is the same, the ACA sequence appears at The predicted frequency in RNA transcripts is 1 / 64. It will be appreciated that some RNA transcripts contain ACA sequences at lower or higher frequencies than predicted frequencies. Thus, the susceptibility of a particular RNA transcript or related family of RNA transcripts to cleavage by the MazF endoribonuclease depends on the frequency of the ACA sequence or the MazF target sequence in the transcript. Furthermore, one of ...

Embodiment III

[0289] As described above, in Escherichia coli, programmed cell death is thought to be mediated by a system of "addicted components", each of which consists of a pair of co-expressed genes encoding a stable toxin and unstable antitoxins. Their expression is autoregulated by the toxin / antitoxin complex or by the antitoxin alone. When coexpression is inhibited, the antitoxin is rapidly degraded by proteases, allowing the toxin to act on its target. In Escherichia coli, extrachromosomal elements are the major genetic system for bacterial programmed cell death. The most studied extrachromosomal addictive component is the phd-doc on phage P1 (Lehnherr et al. (1993) J Mol Biol 233, 414-428; Lehnherr and Yarmolinsky (1995) Proc Natl Acad Sci USA 92, 3274-3277; Magnuson and Yarmolinsky (1998) J Bacteriol 180, 6342-6351; Gazit and Sauer (1999) J Biol Chem 274, 16813-16818; Gazit and Sauer (1999) J Biol Chem 274, 2652-2657), ccdA-ccdB on F factor ( Tam and Kline (1989) JBacteriol 171...

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Abstract

The present invention is directed to the discovery of a novel family of enzymes designated herein as mRNA interferases that exhibit endoribonuclease activity. The novel finding of the present inventors, therefore, presents new applications for which mRNA interferase nucleic and amino acid sequences, and compositions thereof may be used to advantage. The invention also encompasses screening methods to identify compounds / agents capable of modulating mRNA interferase activity and methods for using such compounds / agents. Also provided is a kit comprising mRNA interferase nucleic and / or amino acid sequences, mRNA interferase activity compatible buffers, and instruction materials.

Description

field of invention [0001] The present invention relates to the field of molecular biology, especially the discovery of novel enzyme activities. In particular, the present invention relates to the identification of a novel family of proteins designated herein as mRNA interferases. Exemplary members of the families described here include MazF and PemK, and their homologs and orthologs. More particularly, the present invention relates to the characterization of the biochemical properties of MazF and PemK polypeptides as endoribonucleases or mRNA interferases. The invention also includes the analysis of related proteins that function to inhibit the activity of mRNA interferase. In particular, the characterization of the function of the MazE protein and its effect on MazF activity, and the characterization of the function of the Peml protein and its effect on the activity of PemK are described here. Also provided herein are methods of use of novel mRNA interferases, such as MazF...

Claims

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Application Information

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IPC IPC(8): C12Q1/68A61K31/7105C12P21/06C12P19/34C12P21/00
Inventor M·伊诺耶J·张Y·L·张
Owner UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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