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Compositions and methods for regulating peptidyltransferase activity and uses thereofRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.)Compositions and methods for regulating peptidyltransferase activity and uses thereof description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050277607, Compositions and methods for regulating peptidyltransferase activity and uses thereof. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] Programmed ribosomal frameshift (PRF) events most commonly induce translating ribosomes to slip by a single base in either the 5' (-1) or 3' (+1) direction, though examples of ribosomal "hops", "shunts", and "bypasses" have also been documented (reviewed in Jacks, 1990; Farabaugh, 1996; Gesteland & Atkins, 1996). Such translational recoding signals have been valuable in addressing questions relating to ribosome structure and function. For viruses that utilize PRF, the efficiencies of frameshift events are critical: they determine the stoichiometry of viral structural to enzymatic proteins available for virus particle assembly, and altering PRF frequencies have dire consequences for virus propagation (reviewed in Dinman et al., 1998). Thus, it is important to understand how frameshifting efficiencies are controlled. The most widespread mechanisms involve inducing ribosomes to stall with their associated tRNAs positioned over specific mRNA sequences called "slippery sites" such that, in the event of slippage, the tRNAs are able to base pair with the out-of-frame codon or codons. Though the cis-acting signals are relatively well characterized, the trans-acting factors and the biophysical parameters that contribute to determine PRF efficiencies are less well understood. Genetic, biochemical, molecular, and pharmacological methods have been employed toward this end. In general, parameters that can affect PRF efficiencies include: 1) changes in the residence time of ribosomes at a particular PRF signal and the precise steps of the elongation cycle that such kinetic changes might occur; 2) changes in the stabilities of ribosome-bound tRNAs and/or ribosome catalytic function due to alterations in intrinsic ribosomal components such as ribosomal proteins, rRNAs, and codon:antidcodon interactions; and 3) defects in the abilities of the translational apparatus to recognize and correct errors (reviewed in Harger et al., 2002). [0003] The genetic manipulability of the yeast Saccharomyces cerevisiae has facilitated the identification of trans-acting factors that can affect frameshifting efficiencies, and researchers in the field have capitalized on the presence of two endogenous viruses of yeast to this end. The Ty1 retrotransposable element of yeast utilizes a programmed +1 frameshift to synthesize its Gag-pol precursor (Clare et al., 1988; Belcourt & Farabaugh, 1990), and changes in +1 PRF efficiencies have inhibitory effects on Tyl retrotransposition frequencies (Xu & Boeke, 1990; Kawakami et al., 1993; Balasundaram et al., 1994; Turner et al., 1998; Harger et al., 2001; Hudak et al., 2001). The 4.6 kb dsRNA L-A virus of yeast utilizes a programmed -1 ribosomal frameshift to produce its Gag-pol fusion protein (Icho & Wickner, 1989; Dinman et al., 1991; Tzeng et al., 1992), and changes in -1 PRF efficiencies promote loss of the killer phenotype as a consequence of loss of the 1.6-1.8 kb dsRNA M.sub.1 satellite virus that encodes the secreted killer toxin (reviewed in Wickner, 1996). It has been previously reported that -1 PRF efficiencies were specifically elevated in cells harboring the mak8-1 allele of RPL3 (Peltz et al., 1999), thus providing an explanation for the original observation that mak8-1 cells could not maintain the killer phenotype (Wickner & Leibowitz, 1974; Wickner et al., 1982). Two alleles of RPL3 have been heretofore described: the tcml-1 allele contains a single missense mutation changing tryptophan at position 255 to cystine (Fried & Warner, 1981), and the mak8-1 allele contains this mutation plus a second missense mutation changing proline at position 257 to threonine. In Example 1 herein, the effects of single and double mutations at this site on -1 PRF, killer virus maintenance, and peptidyltransferase activities in isogenic rpl3 gene deletion strains, are described. A PCR-based mutagenesis approach is employed to identify and characterize a new allele of RPL3 consisting of mutation of isoleucine 282 to threonine (I282T) that was unable to maintain the yeast killer virus. All of the mak8 alleles promoted increased -1 PRF efficiencies, and ribosomes isolated from cells expressing these alleles had decreased peptidyltransferase activities Molecular modeling based on the Haolarcula marismortui 50S ribosomal subunit (Ban et al., 2000) reveals that W255 is the closest amino acid residue in the ribosome to the peptidyltransferase center active site, that P257 is required to form an important bend in a loop that positions W255, and that 1282 is in the hydrophobic core at the base of the loop. How these structural changes might specifically affect peptidyltransferase function and -1 PRF is discussed within the context of the recent explosion of information pertaining to ribosome structure and function. Further, based on a recent study showing that deletion of ribosomal protein L41 results in peptidyltransferase defects, both -1 and +1 PRF in isogenic rpl41-deficient and wild-type strains were assayed as described herein (see, Example 1). The finding that -1 PRF was also specifically stimulated in rpl41-deficient strains provides additional evidence that -1 PRF efficiencies can be influenced by peptidyltransfer rates. SUMMARY OF THE INVENTION [0004] The present invention provides compositions and methods to identify compositions that increase -1 PRF efficiencies, and thus directly affect viral replication or assembly of viral particles. Compositions in accordance with the invention specifically inhibit the interaction between ribosomal protein L41 and the ribosomes thereby resulting in decreased peptidyltransferase activity of the ribosomes. While not intending to be limited to any one scientific theory, it is believed that decreases in peptidyltransferase activity result in increased -1 PRF efficiencies which in turn interfere with self assembly of -1 PRF dependent viruses thereby interfering with virus propagation. BRIEF DESCRIPTION OF THE DRAWINGS [0005] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings. [0006] FIGS. 1A and 1B. The rpl3 mutant alleles cannot propagate the yeast killer virus. A. Killer assay of strains harboring the wild-type RPL3 gene or mutant alleles. Isogenic Killer+strains containing the RPL3::HIS3 gene disruption and harboring the wild-type RPL3 gene on a URA3-CEN6 plasmid were transformed with TRP1-CEN6 plasmids containing either the wild-type gene or the indicated rpl3 alleles. After selection on medium lacking tryptophan, cells having lost the URA3-CEN6 plasmids were identified by their ability to grow on medium containing 5-FOA. Colonies were then replica plated onto a lawn of cells that are sensitive to the secreted killer toxin produced by the M.sub.1 satellite virus of L-A. Killer activity was observed as a zone of growth inhibition around the colonies. mak8-1* is not isogenic with the other strains, but is rather the original mak8-1 isolate (RW1906) used here for comparison. B. Total RNAs isolated from the isogenic strains described in (A) were separated through a 1.5% TAE-agarose gel. L-A and M.sub.1 dsRNAs are indicated as 2.5 and 1.8 kbp bands respectively. Top panel shows the ethidium bromide stained gel and the bottom panel is a Northern blot of the gel probed for the presence of L-A and M.sub.1 (-) strand viral RNAs were performed as previously described (Dinman & Wickner, 1994). [0007] FIGS. 2A, 2B and 2C. Elevated efficiencies of -1 programmed ribosomal frameshifting in cells expressing mutant rpl3. A. Monscistronic PRF reporter plasmid system. Isogenic strains harboring the TRP1-CEN6 based RPL3 alleles were co-transformed with p0 or p-1 (LEU2-CEN6 based vectors), or p0 or p+1 (URA3-CEN6) and programmed ribosomal frameshifting efficiencies were determined as described (Dinman et al., 1991; Peltz et al., 1999). Changes in frameshift efficiencies are depicted in terms of fold wild-type (see Table 2). Each dataset represents the averages of three individual experiments repeated in triplicate. Error bars denote standard deviations from the means. B. Bicistronic PRF reporter plasmid system. Isogenic strains harboring the TRP1-CEN6 based RPL3 alleles were co-transformed with bicistronic URA3-CEN6 based Renilla-firefly luciferase 0-frame, -1 (L-A derived), or +1 (Tyl-derived) reporter vectors. Renilla and firefly luciferase activities of clarified cell lysates were determined using Dual-Luciferase Assay Reagents (Promega) and quantitated using a TD20/20 lumineter (Turner designs). Frameshifting efficiencies were calculated by dividing the firefly/Renilla luminescence ratios from the -1 and +1 programmed frameshift test reporters by the 0-frame control reporter. Each dataset represents the averages of three individual experiments repeated in triplicate. Changes in frameshift efficiencies are depicted in terms of fold wild-type (see Table 2). Error bars denote standard deviations from the means. C. Expression of mutant forms of L3 do not affect the nonsense mediated mRNA decay (NMD) pathway. Total cellular mRNA was extracted from mid-logarithmically growing rpl3.DELTA. cells harboring plasmids encoding the wild-type or mutant alleles of RPL3 as indicated. mRNA from PLY36 cells harboring the upf1.DELTA.::hisG allele (upf1.DELTA.) was used as a control. Total RNA from each sample was separated through a 1.0% agarose formaldehyde denaturing gel and transferred to a nylon membrane. The RNA blot was subsequently hybridized with a radiolabeled CYH2 probe as previously described (Cui et al., 1996). The image was visualized, and band intensities were quantitated using a Molecular Dyanmics phosphorimager. The locations of the NMD-sensitive CYH2-precursor mRNA (pre-CYH2) and of the NMD-insensitive mature CYH2 mRNA are indicated, as are the pre-CYH2/CYH2 signal intensity ratios. [0008] FIG. 3. Ribosomes containing mutant forms of L3 have decreased peptidyltransferase activities. Time course of the formation of [.sup.14C]Phenylalanine-puromycin product in assays using ribosomes isolated from isogenic RPL3::HIS3 cells expressing wild-type or mutant forms of L3. Plasmid-borne RPL3 alleles are indicated. EtOAc soluble radioactivity was determined by liquid scintillation counting. Control studies were performed in the absence of puromycin to determine the nonspecific extraction of CACCA[.sup.14C]AcPhe. Control values (generally less than 2%) were subtracted from the values obtained in the presence of puromycin. All experiments were performed in triplicate. Data points and error bars indicate mean and standard deviation. [0009] FIG. 4. Programmed -1 ribosomal frameshifting is specifically stimulated in L41-deficient yeast cells. Isogenic XY5a strains (MATa ade2-1 trp1-1 his3-11,15 can1-100 ura3-1 leu2-3,112 rpl41a::HIS3 rpl41b::URA3) harboring pRS314-RPL41A or pRS314 were co-transformed with either monocistronic or bicistronic p0, p-1 or p+1 LEU2-CEN6 based reporter vectors. Changes in frameshift efficiencies are depicted in terms of fold wild-type (see Table 2). Programmed ribosomal frameshifting efficiencies using the monocistronic reporters were determined as described (Dinman et al., 1991; Peltz et al., 1998). ND: not determined. In the assays using the bicistronic reporter system, Renilla and firefly luciferase activities of clarified cell lysates were determined using Dual-Luciferase Assay Reagents (Promega) and quantitiated using a TD20/20 lumineter (Turner designs). Frameshifting efficiencies were calculated by dividing the firefly/Renilla luminescence ratios from the -1 and +1 programmed frameshift test reporters by the 0-frame control reporter. Each dataset represents the averages of three individual experiments repeated in triplicate. Error bars denote standard deviations from the means. [0010] FIGS. 5A and 5B. L3 and the peptidyltransferase center. A. ClustalW alignment of selected regions of ribosomal protein L3. The primary amino acid sequences from six representative species in the vicinity of the amino acids of L3 examined in this study were aligned. L3 sequences were from the following species: H. Sapiens (HsL3 from amino acids 247-290), D. melanogaster (DmL3, from amino acids 247-290), S. cerevisiae (ScL3 from residues 244-287), H. marismortui (HaL3 from residues 233-276), E. coli (EcL3 from residues 139-183), and T. thermophilus (TtL3 from amino acids 134-178). The three amino acids of interest to this study are highlighted. B. Is a map of the L3 mutants within in the context of the H. marismortui 50S crystal structure at 2.4 .ANG.. DETAILED DESCRIPTION OF THE INVENTION [0011] In a first aspect, the invention provides methods to identify compositions that inhibit peptidyltransferase activity by inhibiting the interaction of L41 with ribosomes, comprising the steps of: [0012] a) contacting a test composition with a cell or cell extract containing L41 and ribosomes in an amount sufficient to inhibit peptidyltransferase activity; [0013] b) detecting whether the test composition inhibits the interaction of L41 with the ribosomes; and [0014] c) determining whether the test composition inhibits peptidyltransferase activity of the ribosomes. [0015] In one non-limiting example of the first aspect of the invention, a cell-based assay system can be devised, in which recombinant ribosomes that have been modified to include a Streptavidin affinity tag in the 25S ribosomal RNA (rRNA) are expressed in a host cell lacking wild-type L41. Also expressed in the same host cell is a recombinant form of L41 that comprises an affinity tag that can bind to a fluorescently labeled indicator molecule. A test composition is introduced into the host cell for a time sufficient to allow the test compound to interact with the tagged L41 protein, and perhaps bind the protein and prevent it from associating with the ribosomes. The cells are then lysed and the contents contacted with streptavidin beads to which the ribosomes will adhere. The beads are then probed with a fluorescent indicator molecule that corresponds to the affinity tag on the L41 protein. Under normal circumstances, L41 should associate with the ribosomes. However, if the test composition displaces L41 from the ribosome or prevents L41 from interacting with the ribosome, unassociated L41 protein remain part of the supernatant and be will be removed when the beads are washed. Fluorescence detected in the wash corresponds to the tagged L41 that was unable to associate with the ribosomes. Peptidyltransferase activity of the ribosomes can then be tested by eluting the ribosomes from the straptavidin beads (e.g. by the addition of biotin) and assaying for peptidyltransferase activity using for example, the puromycin reaction described in Example 1. [0016] Two non-limiting examples of the first aspect of the invention using mechanism-based assays can also be designed around the tagged ribosomes and L41 proteins. In one form, a test composition is introduced into a reaction mixture composed of affinity tagged ribosomes containing the tagged L41 protein for a time sufficient to allow the test compound to interact with the tagged L41 protein, and dissociate the protein from the ribosomes. In another form, test composition is first introduced into a reaction mixture of affinity tagged ribosomes lacking the L41 protein, and after a sufficient time, recombinant tagged L41 is added. In yet another form, the test composition is first introduced into a reaction mixture of affinity tagged ribosomes lacking the L41 protein, and after a sufficient time, recombinant tagged L41 is added: here the compound would inhibit the ability of L41 to initially associate with the ribosome. In both cases, the compound of interest would interfere with the ability of L41 to interact with the ribosome. The reaction mixtures are then contacted with streptavidin beads to which the ribosomes will adhere. The beads are then probed with a fluorescent indicator molecule that corresponds to the affinity tag on the L41 protein. Under normal circumstances, L41 should associate with the ribosomes. However, if the test composition displaces L41 from the ribosome or prevents L41 from interacting with the ribosome, unassociated L41 protein remain part of the supernatant and be will be removed when the beads are washed. Fluorescence detected in the wash corresponds to the tagged L41 that was unable to associate with the ribosomes. Peptidyltransferase activity of the ribosomes can then be tested by eluting the ribosomes from the straptavidin beads (e.g. by the addition of biotin) and assaying for peptidyltransferase activity for example, the puromycin reaction described in Example 1. [0017] As used herein, the term "inhibiting peptidyltransferase" refers to a reduction or decrease in the level of activity of peptidyltransferase of a ribosome as compared to its normal level of peptidyltransferase activity. As used herein the term "inhibiting the interaction of L41 with ribosomes" refers to any alterations in the normal binding and/or interaction between L41 with the ribosomes such as preventing L41 from associating with the ribosome in the normal manner, or displacing L41 from the ribosome, or otherwise altering or preventing the ribosome to carry out its normal activities if L41 was otherwise normally associated with the ribosome. Any composition can be used as a test composition in practicing the invention; preferred test compositions include but are not limited to, polypeptides and small molecules. Although sequence or structural homology can provide a basis for suspecting that a test composition can modulate the interaction with L41 in a cell, randomly chosen test compositions also are suitable for use in the invention. Art-known methods for randomly generating test compositions (e.g., expression of polypeptides from nucleic acid libraries; combinatorial chemistry techniques for generating small molecule chemical libraries) can be used to produce suitable test agent or compositions. Those skilled in the art will recognize alternative techniques can be used in lieu of the particular techniques described herein. [0018] In one embodiment the cell or cell extract may be a yeast cell, cell extract, or purified components such as those from Saccharomyces cerevisiae. [0019] In accordance with the present invention such as for the assays described herein, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). [0020] In a second aspect, the invention provides methods of determining whether a test composition increases -1 PRF efficiencies of ribosomes by inhibiting the interaction of L41 with the ribosomes, comprising the steps of: [0021] a) contacting a test composition with a cell or cell extract or purified components mixture containing L41 and ribosomes; Continue reading about Compositions and methods for regulating peptidyltransferase activity and uses thereof... Full patent description for Compositions and methods for regulating peptidyltransferase activity and uses thereof Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compositions and methods for regulating peptidyltransferase activity and uses thereof patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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