CA2341765A1 - Trna binding domain - Google Patents

Abstract

There is disclosed a substantially pure peptide comprising the following sequence: [ML] -X(3) -[LIVMC] -X(3) -[GMNEHK] -[MLFYI] -X(2) -[ST] -X(3) - [SAIG] - [RMIK] -X(2) -[MIVA] -X(2) -[GNRHK] -X-[IVLF] -X-[LIV] -[NDSRGAL] - X(3) -[LIVSQ] -X(2) -[PILVTAC] wherein X represents any amino acid. Also disclosed is a method for determining whether a nucleic acid comprises a tRN A anticodon stem-loop. Another aspect comprises a method for identifying a substance which binds to a wherein the binding is detected by assaying for conjugates, for free substance, or for non-complexed peptide. Also described is a method of determining whether a test compound is an agonist or antagoni st of a tRNA synthetase-tRNA anticodon stem-loop interaction or ribosomal S4 protein-tRNA anticodon stem-loop interaction. There is also described a meth od for obtaining a substantially pure nucleic acid comprising a tRNA anticodon stem-loop from a mixture of different nucleic acids. Finally, there is described a pharmaceutical composition for inhibiting the interaction of a tRNA, with a tRNA synthetase or ribosomal protein along with other related aspects.

Description

TITLE: NOVEL tRNA BINDING DOMAIN
FIELD OF THE INVENTION
The invention relates to a novel domain that binds to tRNA; peptides derived from the domain which modulate the interaction of tRNA with proteins including tRNA
synthetases and ribosomal 5 proteins; and, uses of the peptides. The invention also relates to complexes comprising a domain or peptide of the invention and a tRNA or portion thereof.
BACKGROUND OF THE INVENTION
A number of important cellular activities are carried out by protein-tRNA
complexes. The recognition of tRNA by their cognate aminoacyl-tRNA synthetases is the critical step in the translation 10 of the genetic code. The tRNA synthetases catalyze the aminoacylation of tRNA in a two-step reaction.
The amino acid is first activated with ATP to form an amino acid adenylate and pyrophosphate, then the adenylate is attacked by the 3'terminal ribose of the tRNA to form amino acid-tRNA and AMP.
Ribosomes interact with aminoacyl tRNA, peptidyl tRNA, and exiting tRNA, and these interactions account for the codon-anticodon interaction between mRNA and tRNA, the correct 15 positioning of tRNA acceptor and donor arms during peptide bond formation, and the movement of mRNA relative to the ribosome. Ribosomal protein S4 is a multifunctional protein associated with the 30S subunit, comprising 206 amino acids in E. toll, which has been implicated in the binding of aminoacyl tRNA.
Inhibition of tRNA protein interactions leads to a reduction in protein translation triggering a 20 cascade of responses. For example in prokaryotes, inhibition of tRNA
synthetases may lead to a state of dormancy in the organism. Therefore, selective inhibitors of bacterial or fungal protein biosynthesis have potential as antibacterial agents. Inhibitors are also potentially useful as anti-viral agents, immunotoxins, and plant toxins.
SUMMARY OF THE INVENTION
25 The present inventor has identified a novel domain within the C-terminus of a tyrosyl-tRNA
synthetase and the ribosomal S4 protein that mediates the binding of the proteins to a tRNA anticodon stem-loop.
Broadly stated the present invention provides a novel domain and peptides derived therefrom having the following sequence:

[MLJ-X(3)-[LIVMCJ-X(3)-[GMNEHK]-[MLFYI]-X(2)-[ST]-X(3)-[SAIG]-[RMIK]-X(2)-[MIVA]-X(2~[GNRHK]-X-[IVLF]-X-[LIV]-[NDSRGAL]-X(3)-[LIVSQ]-X(2)-[PILVTAC]
wherein X represents any amino acid.
35 In accordance with an embodiment of the invention peptides are provided comprising the sequence motif Y,-X(3)-YZ-X(3)-Y3-Y4-X(2)-YS-X(3)-Y6-Y7-X(2)-Y8-X(2)-Y9-X-Y,o-X-Y"-Y,2-X(3)_ Y,3-X(2~Y,4 where Y, is methionine or leucine, preferably leucine, YZ is leucine, isoleucine, valine, methionine, or cysteine, preferably isoleucine, leucine, or valine,Y3 is glycine, methionine, asparagine, glutamic acid, histidine, or lysine, preferably glycine, Y4 is methionine, leucine, phenylalanine, 40 tyrosine, or isoleucine, preferably phenylalanine, leucine, methionine, or tyrosine, YS is serine or WO 00/11141 PCTlCA99/00779 threonine, Y6 is serine, alanine, isoleucine, or glycine, preferably alanine, Y~ is arginine, methionine, isoleucine, or lysine, preferably arginine, Y8 is methionine, isoleucine, valine, alanine, preferably valine or isoleucine, Y9 is glycine, asparagine, arginine, histidine, or lysine, preferably lysine, glycine, or arginine, Y,o is isoleucine, valine, leucine, or phenylalanine, preferably valine or isoleucine, Y" is leucine, isoleucine, or valine, preferably valine or isoleucine, Y,Z is asparagine, aspartic acid, serine, arginine, glycine, alanine, or leucine, preferably asparagine, aspartic acid, or glycine,Y~3 is leucine, isoleucine, valine, serine, or glutamine, preferably glutamine or valine, Y,4 is proline, isoleucine, leucine, valine, threonine, alanine, cysteine, or serine, preferably proline or valine, and X is any amino acid.
10 The invention also provides biologically, diagnostically, prophylactically, clinically, or therapeutically useful variants thereof, and compositions comprising the peptides and variants. In particular, the invention contemplates truncations and analogs of the peptides of the invention.
The present invention also relates to a complex comprising a peptide having the following sequence motif:

[ML]-X(3r[LIVMC]-X(3)-[GMNEHK]-[MLFYI]-X(2)-[STJ-X(3)-[SAIG]-[RMIK]-X(2)-[MIVAJ-X(2)-[GNRHKJ-X-[IVLF]-X-[LIV]-[NDSRGALJ-X(3)-[LIVSQ]-X(2Jh[PILVTAC]
wherein X represents any amino acid, with a tRNA anticodon stem-loop.
20 The invention also contemplates antibodies specific for the complexes and peptides of the invention.
The invention also relates to the use of a peptide or complex of the invention to interfere with the interaction of a tRNA anticodon stem-loop (e.g. tRNA), with proteins comprising a domain of the invention including tRNA synthetases or ribosomal proteins and, pharmaceutical compositions for 25 inhibiting the interaction of a tRNA anticodon stem-loop (e.g. tRNA), with proteins including tRNA
synthetases or ribosomal proteins. The peptides, compositions and antibodies may be used to interfere with protein synthesis and they may be used as antibacterial agents, anti-viral agents, immunotoxins, or plant toxins.
Further, the invention relates to a method of modulating protein synthesis and in particular the 30 interaction of a tRNA anticodon stem-loop (e.g. tRNA), with a tRNA
synthetase or ribosomal protein comprising changing the following sequence motif in a tRNA synthetase or ribosomal protein:
[ML]-X(3)-[LIVMC]-X(3)-[GMNEHK]-[MLFYI]-X(2r[ST]-X(3)-[SAIG]-[RMIK]-X(2}-[MIVA]-X(2)-[GNRHK]-X-[IVLF]-X-[LIV]-[NDSRGAL]-X(3)-[LIVSQ]-X(2)-[PILVTAC], wherein X
is any 35 amino acid.
The present invention also provides a method for determining whether a nucleic acid comprises a tRNA anticodon stem-loop. The method comprises the steps of contacting a nucleic acid with a peptide of the invention and determining whether the peptide binds to the nucleic acid. The binding of the peptide to the nucleic acid is indicative that the nucleic acid comprises a tRNA
anticodon stem-loop.
In another embodiment, the present invention provides a method of determining whether a test compound is an agonist or antagonist of a protein comprising a domain of the invention (i.e. a tRNA
5 anticodon stem-loop recognition motif) and a tRNA anticodon stem-loop, including a tRNA
synthetase-tRNA anticodon stem-loop interaction or ribosomal S4 protein-tRNA
anticodon stem-loop interaction. The method comprises the steps of incubating the test compound with a nucleic acid comprising a tRNA anticodon stem-loop, and a peptide of the invention, determining the amount of nucleic acid bound to the peptide during the incubating step, and comparing the amount of nucleic acid 10 bound to the peptide during the incubating step to an amount of nucleic acid bound to peptide in the absence of the test compound. An increase in the amount of nucleic acid bound to peptide in the presence of the test compound will be indicative that the test compound is an agonist of an interaction, while a decrease indicates that the test compound is an antagonist of an interaction.
In an additional embodiment, a method is provided for obtaining a substantially pure nucleic 15 acid comprising a tRNA anticodon stem-loop from a mixture of different nucleic acids. The method comprises the steps of providing a peptide of the invention bound to a solid support. The mixture of different nucleic acids is contacted with the peptide bound to the solid support whereby a nucleic acid comprising a tRNA anticodon stem-loop is bound to the peptide. The solid support is washed to remove unbound nucleic acids and substantially pure nucleic acids comprising a tRNA anticodon stem-20 loop are then eluted from the solid support.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention 25 will become apparent to those skilled in the art from this detailed description.
DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to the drawings in which:
Figure 1. The hydrophobic motif shared between ribosomal S4 protein and Tyrosyl-tRNA
synthetase. Yellow residues comprise the hydrophobic core. An invariant Ser/Thr residue shown in 30 green delimits the N-terminal end of a common central helix. Arginine residues shown in the B.
stearothermophilus TyrRS (red blocks) were shown to bind to tRNATn through site-directed mutagenesis Figure 2. Threading alignment of Synechocystis TyrRS residues 342-404 and the fragment 94-159 from the B. stearoihermophilus S4 3-D. Boxed regions are aligned by the procedure and 35 identical residues are shown in bold. A suboptimal alignment in the threading ensemble has an altered alignment of the third block that closes both gaps, but it has a slightly lower Z score. Starting with a 3-D core comprising 38% of this structure, shown with the wavy lines, the final threading alignment recruited a total of 80% of residues in the S4 structure fragment with a Z-score of 4.55. The self thread recruited 91% of residues with a Z-score of 6.3. Randomized sequences with the same composition 40 made poor alignments with very many alternative suboptimal alignments.

WO 00)11141 PCT/CA99/00779 Figure 3. Cartoon image of the B. stearothermophilus ribosomal S4 structure fragment showing the TyrRS similarities. The blue filled backbone cartoon corresponds to the motif shown in Figure 1. The entire blue and pink filled backbone cartoon represents the substructure that was used in the threading analysis. The backbone drawn with pink lines corresponds to the N-terminus. The grey 5 lines correspond to the C-terminus of S4 that is not in common with TyrRS.
This fragment is completely missing in the archeal S4 proteins, and may not be structurally required to form the motif fold shown in blue/pink. Yellow residues represent the hydrophobic core and correspond to yellow residues in the motif shown in Figure 1. Blue residues represent basic side chains that align with those of TyrRS previously shown to interact with tRNATn . The green residue shows the position of the 10 conserved threonine/serine in the motif as a helical N-cap. The helix at the top left of the structure (pink lines) corresponds to the site of ram mutations, while the helix at the bottom right (solid pink) corresponds to the Ets-domain DNA binding helix similarity.
Figure 4. Neighbor joining clustering using ClustalX of the motif in Figure 1.
Information in this motif is sufficient to reconstruct, from bottom to top, clusters corresponding to archea, eukaryotes, 15 chloroplasts and their photosynthetic relatives, and eubacteria.
Mitochondria) S4 sequences and TyrRS
sequences diverge from the cluster in an atypical fashion, suggesting a change in evolutionary rates.
This divergence may correspond to a systematic decrease in the population of cognate tRNA from >20 to one or two in the case of TyrRS, together with a mixing of cytoplasmic and mitochondria) tRNAs for use in the mitochondrion.

Glossary The following standard abbreviations for the amino acid residues are used throughout the specification: A, Ala - alanine; C, Cys - cysteine; D, Asp- aspartic acid; E, Glu - glutamic acid; F, Phe -phenylalanine; G, Gly - glycine; H, His - histidine; I, Ile - isoleucine; K, Lys - lysine; L, Leu - leucine;
25 M, Met - methionine; N, Asn - asparagine; P, Pro - proline; Q, Gln -glutamine; R, Arg - arginine; S, Ser - serine; T, Thr - threonine; V, Val - valine; W, Trp- tryptophan; Y, Tyr -tyrosine; and p.Y., P.Tyr - phosphotyrosine.
The amino acids used in the peptides of the invention are preferably in the "L" isomeric form.
However, stereoisomers (e.g. D-amino acids) of the twenty conventional amino acids, unnatural amino 30 acids such as a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for peptides of the present invention. Examples of unconventional or unnatural amino acids include amino acids well known in the art, but which are not included in the twenty conventional amino acids, such as 3 or 4-hydroxyproline, y-carboxyglutamate, e-N,N,N-trimethyllysine, E-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-35 methylhistidine, 5-hydroxylysine, w-N-methylarginine, and other similar amino acids and imino acids.
In the peptide notation used herein, the IeRhand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
T'he term "protein comprising a tRNA anticodon stem-loop recognition motif' refers to a 40 protein comprising the following sequence motif [ML]-X(3)-[LIVMC]-X(3}-[GMNEHK]-[MLFYIJ-X(2)-[ST)-X(3r[SAIG]-[RMIKJ-X(2)-[MIVA]-X(2)-[GNRHK]-X-[IVLF]-X-[LIVJ-[NDSRGAL]-X(3~[LIVSQ]-X(2)~[PILVTAC], wherein X
is any amino acid. Examples of the proteins are tRNA synthetases, and ribosomal proteins.
The tenor "tRNA synthetase" refers to a protein or peptide which comprises or consists of a sequence which is capable of binding to a tRNA anticodon stem-loop. Examples of tRNA synthetases are tyrosyl tRNA synthetase [see for example Guez-Ivanier and Bedouelle (25), Brick et al. (2) and Brick and Blow (I ) re the structure of tyrosyl-tRNA synthetase from B.
stearothermophilus;
W09739015, and W09726351 j; isoleucyl tRNA synthetase (Chalker, A. F. et al Gene 141:103, 1994);
10 valyl tRNA synthetase (LJ.S. Nos. 5,789,218, and 5,747,314, W09726355, EP0785267); asparaginyl tRNA synthetase (U.S. 5,789,21, W09726348); alanyl tRNA synthetase (U.S.
5,776,750, W09739013, W09726353); cysteinyl tRNA synthetase (LJ.S. 5,775,749, U.S. 5,753,480, W09726341); arginyl tRNA synthetase (U.S. 5,763,246, W09726347, EP0785266); glycyi tRNA synthetase (U.S.
5,756,330, W09726340); phenylalanyl tRNA synthetase (U.S. 5,756,329, U.S.
5,753,479, 15 W09726356); tryptophanyl tRNA synthetase (EP0843014); leucyl tRNA
synthetase (LJS. 5,750,387, W09726349); histidyl tRNA synthetase (iJ.S. 5,747,313, W09739017, W09726354, EP0785269);
seryl tRNA synthetase (U.S. 5,744,338, W09726352, EP0785270); threonyl tRNA
synthetase (EP0815237, W0972634, EP0785271); aspartyl tRNA synthetase (U.S. 5,747,315, W09739014, W09726344); methionyl tRNA synthetase (W09726350, W09739012, EP0785268), isoleucyl tRNA
20 synthetase (W09739011); tryptophanyl tRNA synthetase (W09726346); glutamyl tRNA synthetase (WO9726345); and propyl tRNA synthetase (W09726343, EP0785272). tRNA
synthetases may also be identified by using antibodies or probes specific for the enzymes.
The term "ribosomal protein" refers to a ribosomal protein or peptide that comprises or consists of a sequence that is capable of binding tRNA and in particular binding a tRNA anticodon 25 stem-loop. Preferably the ribosomal protein is ribosomal S4 protein which is a multifunctional ribosomal protein associated with the 30S subunit, comprising 206 amino acids in E. toll. S4 is an ancient, if not one of the most ancient of ribosomal proteins (5). In the ribosome, S4 is required and it is the fu~st protein involved in the folding of 16S rRNA. (7) The term includes mutations of S4 proteins, specifically the ram (ribosomal ambiguity) D14 and D12 mutants in E. toll (8,9,10), the omnipotent 30 suppressor mutant SUP46 in yeast ( 11 ), and the NAM-9 mutants of yeast ( 12).
The term "tRNA anticodon stem-loop " refers to a nucleic acid comprising or consisting of an anticodon sequence of a tRNA or a chemically, enzymatically, or metabolically modified form thereof, which has high affinity to a domain of the invention and proteins comprising such a domain including tRNA-synthetase or ribosomal S4 protein. tRNA anticodon stem-loops may be identified using data 35 base search methods (see for example, www.molgen.uc.edu/analyze/Stem.htm~
http://mell.angis.org.au/Stadenn. tRNA anticodon stem-loops may also be identified by screening libraries with a protein containing a sequence with high affinity to a tRNA
anticodon stem-loop, i.e. a peptide of the invention which may be labeled.

The phrase "interfere with the interaction oP' refers to the ability of the peptides or complexes of the invention to inhibit the interaction of a protein such as a tRNA
synthetase or ribosomal protein and a tRNA anticodon stem-loop thereby affecting protein synthesis.
The term "peptide" refers to macromolecules which comprise a multiplicity of amino or imino acids {or their equivalents) in peptide linkage, wherein the peptides may comprise or lack post-translational modifications (e.g. glycosylation, cleavage, phosphorylation, side-chain derivation and the like).
The terms "label" or "labeled" refer to incorporation of a detectable substance e.g. by incorporation of a radiolabeled amino acid or attachment of biotinyl moieties to a protein or peptide 10 wherein the attached biotinyl moieties can be detected by marked avidin.
Various methods of labeling proteins and peptides are known in the art and may be used. Examples of labels include, but are not limited to the following: radioisotopes (e.g.'H, 14C,'sS,'uI, "'I), fluorescent labels (e.g. FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g. horseradish peroxidase, ~i-galactosidase, luciferase, alkaline phophatase), biotinyl groups, predetermined polypeptide epitopes recognized by a 15 secondary reporter (e.g, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
The term "substantially pure" means that the particular peptide is the predominant species present (i.e. on a weight/volume percentage, it is the most abundant single species within the 20 composition), and preferably a substantially purified fraction is a composition wherein the peptide comprises at least about 50 percent (w/v) of all macromolecular species present. Generally, a substantially pure composition will comprise more than 80 to 90 percent of all protein present in the composition. Most preferably, the peptide is purified to essential homogeneity (contaminant proteins cannot be detected in the composition by conventional detection methods) wherein the composition 25 consists essentially of a single protein species.
Peptides and Complexes The peptides of the invention generally comprise a core sequence which corresponds to a tltNA anticodon stem-loop recognition sequence motif. This general motif can be identified by the data described herein. Typically, the peptides will comprise the sequence motif Y,-X(3rYZ-X(3)-Y3-Y4-30 X(2)-Ys-X(3)-Y6-YrX(2~Yg-X(2)-Y9-X-Y,o-X-Y"-Y,2-X(3)-Y,3-X(2)-Y,4 where Y, is methionine or leucine, preferably leucine, YZ is leucine, isoleucine, valine, methionine, or cysteine, preferably isoleucine, leucine, or valine,Y3 is glycine, methionine, asparagine, glutamic acid, histidine, or lysine, preferably glycine, Y4 is methionine, leucine, phenylalanine, tyrosine, or isoleucine, preferably phenylalanine, leucine, methionine, or tyrosine, YS is serine or threonine, Y6 is serine, alanine, 35 isoleucine, or glycine, preferably alanine, Y7 is arginine, methionine, isoleucine, or lysine, preferably arginine, Yg is methionine, isoleucine, valine, or alanine, preferably valine or isoleucine, Y9 is glycine, asparagine, arginine, histidine, or lysine, preferably lysine, glycine, or arginine, Y,o is isoleucine, valine, leucine, or phenylalanine, preferably valine or isoleucine, Y" is leucine, isoleucine, or valine, preferably valine or isoleucine, Y,Z is asparagine, aspartic acid, serine, arginine, glycine, alanine, or 40 leucine, preferably asparagine, aspartic acid, or glycine,Y,3 is leucine, isoleucine, valise, serine, or _7_ glutamine, preferably glutamine or valine, Y" is proline, isoleucine, leucine, valine, threonine, alanine, cysteine, or serine, preferably proline or vaiine, and X is any amino acid.
Generally the sequence recognition motif may be present as its own peptide, or may be a core of a longer sequence. Generally, the peptides of the invention will comprise the motif as a portion, or a whole of a peptide of from 10 to about 200 amino acids in length. Typically the peptides will be from about 20 to 100 amino acids in length, preferably the peptides will be from about 30 to about 75 amino acids in length, more preferably from about 36 to about 65 amino acids in length, A peptide of the invention is also represented herein as comprising the following sequence:
10 [ML]-X{3~[LIVMC]-X(3~[GMNEI-IK]-[MLFYI]-X(2~[ST]-X(3r[SAIG]-[RMIK]-X(2~[MIVA]-X(2~[GNRHK]-X-[IVLF]-X-[LIV]-[NDSRGAL]-X(3~[LIVSQ]-X{2)-[PILVTAC]
wherein X represents any amino acid.
The invention also provides complexes comprising a peptide of the invention and a tRNA
15 anticodon stem-loop.
Preferred peptides of the invention include the peptides shown in Figure 1 (SEQ.ID.NOs. l-45) and Figure 2 (SEQ.ID.NO. 46-47). Additional peptides within the scope of the invention may be identified using the sequence set out above for example, with the ScanProsite service at http://expasy.hcuge.ch/sprot/scnpsit2.html.
20 In addition to full-length peptides of the invention, truncations of the peptides which inhibit interaction of a tRNA synthetase or ribosomal protein and a tRNA anticodon stem loop are contemplated in the present invention. Truncated peptides may comprise peptides of about 7 to 10 amino acid residues.
The truncated peptides may have an amino group (-NH2), a hydrophobic group (for example, 25 carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenyhnethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end. The truncated peptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal 30 end.
The peptides of the invention may also include analogs, and/or truncations thereof, which may include, but are not limited to the peptide of the invention containing one or more amino acid insertions, additions, or deletions, or both. Analogs of the peptide of the invention exhibit the activity characteristic of a peptide of the invention (e.g. interference with the interaction of a tyrosyl-tRNA
35 synthetase or ribosomal S4 protein and a tRNA anticodon stem-loop), and may further possess additional advantageous features such as increased bioavailability, stability, or reduced host immune recognition.
One or more amino acid insertions may be introduced into a peptide of the invention. Amino acid insertions may consist of a single amino acid residue or sequential amino acids. One or more _g_ amino acids, preferably one to five amino acids, may be added to the right or left termini of a peptide of the invention.
Deletions may consist of the removal of one or more amino acids, or discrete portions from the peptide sequence. The deleted amino acids may or may not be contiguous.
The lower limit length 5 of the resulting analog with a deletion mutation is about 7 amino acids.
Cyclic derivatives of the peptides of the invention are also part of the present invention.
Cyclization may allow the peptide to assume a more favorable conformation for association with a tRNA anticodon stem-loop. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free 10 sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc.
1995, 117, 8466-8467. The side chains of P.Tyr and Asn may be linked to form cyclic peptides. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two.
15 It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and tum.
The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of 20 hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.
In addition to the above peptides, peptide analogs are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These non-peptide compounds are referred to as "peptide mimetics" or 25 "peptidomimetics" (Fauchere, J. 1986, Adv. Drug. Res. 15:29; Veber and Freidinger 1985, TINS, 392;
and Evans et al 1987, J. Med. Chem. 30:1229). Peptide mimetics are generally developed using computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptide mimetics are structurally similar to a paradigm peptide (i.e, a peptide that has a biological or 30 pharmacological activity) such as a naturally occurring polypeptide (e.g. a tRNA anticodon stem-loop recognition motif of tyrosyl-tRNA synthetase), but have one or more peptide linkages optionally replaced by a linkage from for example a group comprising: -HiNH-, -CHZS-, -CHZCHz-, -CH=CH-(cis and trans), -COCHz-, -CH(OH)CHZ-, and -CHZSO-, by methods known in the art (see for example Spatola, A.F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, 35 eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J.S., Trends Pharm. Sci. (1980), pp.
463-468 (general review); Hudson, D. et al, Int. J. Pept Prot Res. (1979), 14:177-185 (-CHzNH-, CHZCHZ-); Spatola, A.F. et al., Life Sci. (1986), 38:1243-1249 (-CHZ-S); Hann, M.M., J. Chem Soc.
Perkin Trans I (1982), 307-314 (-CH-CH-, cis and traps); Almquist, R.G. et al., J. Med Chem (1980), 40 23:1392-1398 (-COCHZ-); Jennings-White, C. et al., Tetrahedron Lett. 1982, 23:2533 (-COCHZ-);

Szelke, M. et al., European Appln. EP 45665 (1982), CA: 97:39405, (1982) (-CH(OH~HZ-); Holladay, M.W. et al., Tetrahedron Lett. (1983), 24:4401-4404 (-C(OH)CHZ-); and Hruby, V.J., Life Sci. (1982) 31:189-199 (-CH2S-).
Peptide mimetics can have advantages including for example more economical production, 5 greater chemical stability, enhanced pharmacological properties (half life, absorption, potency, efficacy, etc.), altered specificity (e.g. broad spectrum biological activities), and reduced antigenicity.
Labels can be directly or indirectly attached (e.g. through a spacer such as an amide group) to non-interfering positions on a peptide mimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Non-interfering positions typically are positions that do not form direct 10 contacts with the macromolecules to which the mimetic binds to produce the effect. Labeling of mimetics should not substantially interfere with the desired biological or pharmacological activity of the mimetic. Generally, mimetics of the peptides of the invention can bind to a tltNA anticodon stem-loop with high affinity and possess detectable biological activity i.e. are agonists or antagonists to one or more tRNA anticodon stem-loop mediated phenotypes.
15 More stable peptides can be generated by systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid or the same type.
The invention also includes a peptide conjugated with a selected protein, or a detectable substance, or selectable marker (see below] to produce fusion proteins. A
"fusion protein" generally refers to a composite protein made up of two or more separate proteins which are normally not fused 20 together as a single protein. Fusion proteins can be made by either recombinant nucleic acid methods or by chemical synthesis methods well known in the art. Fusion partners can include a substrate, cofactor, inhibitor, affinity ligand, antibody binding epitope tag, or an enzyme capable of being assayed. A
fusion partner can include for example bacterial ~i-galactosidase, trpE, protein A, (3-lactamase, a-amylase, alcohol dehydrogenase, and yeast a-mating factor. Because of their ability to recognize and 25 bind specific proteins e.g. a protein comprising a tltNA anticodon stem-loop, the peptides of the invention may act as an affinity ligand to direct the activity of a fused protein to the specific proteins.
The peptides of the invention may be free in solution or covalently attached to a solid support.
Peptides attached to a solid support can be particularly useful in screening and purification applications. Examples of solid supports include those well known in the art such as cellulose, agarose, 30 polystyrene, divinylbenzene, and the like. Commercially available supports that come prepared for immediate coupling of affinity ligands can be used (e.g. from Sigma Chemical, St. Louis, Missouri, or Pharmacia, Uppsala, Sweden).
The peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or 35 organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.
The peptides of the invention may be prepared using recombinant DNA methods.
Accordingly, nucleic acid molecules which encode a peptide of the invention may be incorporated in a 40 known manner into an appropriate expression vector which ensures good expression of the peptide.

Possible expression vectors include but are not limited to chromosomal, episomal, and virus-derived vectors such as vectors derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, 5 pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, or modified viruses so long as the vector is compatible with the host cell used. The expression vectors contain a nucleic acid molecule encoding a peptide of the invention and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may 10 be obtained from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Other sequences, such as an origin of replication, additional DNA restriction 15 sites, enhancers, and sequences conferring inducibility of transcription may also be incorporated into the expression vector.
The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transfonmed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as 6418 and hygromycin which confer resistance to certain drugs, (3-20 galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.
The recombinant expression vectors may also contain genes which encode a fusion portion which provides increased expression of the recombinant peptide; increased solubility of the 25 recombinant peptide; and/or aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be inserted in the recombinant peptide to allow separation of the recombinant peptide from the fusion portion after purification of the fusion protein. Examples of fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) 30 which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
Recombinant expression vectors may be introduced into host cells to produce a transformant host cell. Transformant host cells include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms 35 "transfonmed with", "transfected with", "transformation" and "transfection"
are intended to include the introduction of nucleic acid (e.g. a vector) into a cell by one of many techniques known in the art. For example, prokaryotic cells can be transformed with nucleic acid by electroporation or calcium-chloride mediated transformation. Nucleic acid can be introduced into mammalian cells using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated 40 transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and _11_ transfecting host cells may be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press ( 1989)), and other laboratory textbooks.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the peptides of the invention may be expressed in bacterial cells such as Streptococci, Staphylococci, Streptomyces, B. Subtilus, E. coli, fungal cells such as yeast cells, insect cells such as Drosophila (using baculovirus), or mammalian cells such as CHO, COS, HeLa, C
127, BHK, 293, and plant cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
In an embodiment of the invention the host cells are plant cells and the vectors which are used 10 to transform the plant tissue include Agrobacterium vectors and ballistic vectors.
The peptides of the invention may be tyrosine phosphorylated using conventional methods including the method described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992). For example, tyrosine phosphorylation may be induced by infecting bacteria harbouring a plasmid containing a nucleotide sequence encoding a peptide of the invention, with a ~,gtl 1 bacteriophage encoding the 15 cytoplasmic domain of the Elk tyrosine kinase as a LacZ-EIlc fusion.
Bacteria containing the plasmid and bacteriophage as a lysogen are isolated. Following induction of the lysogen, the expressed peptide becomes phosphorylated by the Elk tyrosine kinase.
The peptides of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem.
20 Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). By way of example, the peptides may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphotyrosine as the N-fluorenylmethoxy-carbonyl-O-dimethyl phosphono-L-tyrosine derivative.
25 N-terminal or C-terminal fusion proteins comprising a peptide of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the peptide fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins 30 include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.
Antibodies The peptides and complexes of the invention may be used to prepare antibodies immunospecific for such peptides or complexes. Antibodies include monoclonal and polycional antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, and Fab fragments 35 including the products of an Fab immunoglobulin expression library.
Conventional methods can be used to prepare the antibodies. As discussed below, the antibodies may be used to identify proteins containing tRNA anticodon stem-loop recognition motifs.
Screening for tItNA anticodon stem loop recognition motifs and tRNA anticodon stem-loops.
The peptides, antibodies specific for the peptides, and complexes of the invention may be 40 labeled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan. Labeled antibodies specific for the peptides of the invention may be used to screen for tltNA anticodon stem-loop recognition motifs or binding sites in proteins such as tliNA synthetases or ribosomal proteins, and labeled peptides of the invention may be used to screen for tIZIVA anticodon stem-loops.
In an embodiment of the invention, the peptides of the present invention can be used as probes to identify nucleic acids comprising tltNA anticodon stem-loops. The methods allow for the identification of nucleic acids that are specifically involved in protein translation.
Therefore, in one aspect, the peptides of the invention can be used to determine whether a 10 particular nucleic acid comprises a tltNA anticodon stem-loop.
Determination of whether a nucleic acid comprises a tRNA anticodon stem-loop may be carried out using a variety of means. For example, the nucleic acid to be tested can be immobilized on a solid support e.g. a microtiter well, or nitrocellulose membrane. After blocking the remaining groups on the support, the nucleic acid to be tested can be exposed to an appropriate amount of a labeled peptide of the invention.
Detection of label bound to the 15 test nucleic acid indicates that the nucleic acid contains a tltNA
anticodon stem-loop.
In a preferred embodiment, the nucleic acid is attached to a solid support prior to contacting the nucleic acid with a peptide of the invention and the peptide used in the contacting step further comprises a detectable substance. The determining step comprises assaying for the presence of the detectable substance. Alternatively, the peptide of the invention can be attached to a solid support prior 20 to contacting the nucleic acid with the peptide of the invention.
As an affinity ligand the peptides of the invention can be used to purify nucleic acids which comprise a tRNA anticodon stem-loop from a mixture of nucleic acids. Affinity purification of such nucleic acids can be carried out using conventional affinity purification methods well known in the art.
For example, a peptide of the invention can be attached to a solid support as described herein. A
25 mixture of nucleic acids can then be contacted with the peptide bound to the solid support, such that the peptide selectively binds tltNA anticodon stem-loop containing nucleic acids present in the mixture.
The bound nucleic acids can be washed to eliminate unbound nucleic acids.
Substantially pure tIZNA
anticodon stem-loop containing nucleic acids can be eluted from the solid support by conventional elution protocols.
30 The invention broadly provides methods for identifying substances that bind to a peptide or complex of the invention. The invention also contemplates methods for identifying compounds that bind to substances that interact with a complex or peptide of the invention.
Conventional methods such as co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns may be used to identify such substances and compounds. Substances and compounds 35 identified using the methods of the invention may be isolated and characterized (e.g. sequenced) using conventional techniques.
Substances which can bind with a peptide or complex of the invention can be identified by reacting a peptide or complex of the invention with a test substance which potentially binds to the peptide or complex, under conditions which permit the formation of substance-peptide or substance-40 complex conjugates, and removing and/or detecting the conjugates. The conjugates can be detected by assaying for substance-peptide or substance-complex conjugates, for free substance, or for non-complexed peptide or complexes. Conditions which permit the formation of conjugates may be selected having regard to factors such as the nature and amounts of the substance. The conjugates, free substance or non-complexed peptides or complexes may be isolated by conventional isolation S techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against a peptide or complex may be utilized. The antibodies, peptides, complexes, or substances may be labeled with a detectable substance, or they may be bound to a solid support as described herein.
10 X-ray crystallographic studies may be used as a means of evaluating interactions. For example, purified molecules in a conjugate when crystallized in a suitable form are amenable to detection of infra-molecular interactions by x-ray crystallography.
Spectroscopy may also be used to detect interactions and in particular, Q-TOF instrumentation may be used. In addition, two-hybrid systems may be used to detect protein interactions in vivo.
15 Screening Methods The invention also enables screening of compounds that enhance (agonist) or diminish (antagonist) the level of interaction of a protein comprising a tltNA
anticodon stem-loop recognition motif (e.g. tRNA synthetase or ribosomal protein) and a tltNA anticodon stem-loop. The terms "agonist" and "antagonist" as used herein do not imply a particular mechanism of function.
20 In one aspect of the invention to screen for agonists or antagonists, a synthetic reaction mixture, a cellular compartment (e.g membrane, cell envelope, cell wall), or a preparation thereof, comprising a peptide of the invention and a tItNA anticodon stem-loop is incubated in the presence or the absence of a test compound which may be an agonist or antagonist. The ability of the test compound to enhance or interfere can be reflected in increased or decreased binding of the peptide and 25 the tIZNA anticodon stem-loop. The efficiency of the reaction may be enhanced by labeling the peptide or tRNA anticodon stem-loop, using a reporter system, or immobilizing the peptide or tRNA anticodon stem loop upon a solid support.
As a specific example, a nucleic acid comprising a tltNA anticodon stem loop can be coupled to the wells of a microtiter plate or nitrocellulose membrane. The test compound can be added to the 30 well or membrane to preincubate with the nucleic acid. The peptide of the invention, to which a detectable substance is attached is added to the well or membrane. Following sufficient incubation, the wells or membranes are rinsed, and binding of the peptide to the nucleic acid can be assessed by for example assaying for the presence of residual detectable substance. Those of skill in the art will recognize that the screening assay format can be set up in either direction, i.e. either the peptide or 35 nucleic acid can be bound to the support, while the other is labeled. The level of binding can be compared to suitable positive and negative controls. Alternatively, by providing the nucleic acid and/or the peptide in known concentrations, one can assay for free, or unbound nucleic acids and/or peptide, and by negative implication, determine the level of complexes that are formed.
The amount or concentration of the test compound that is added, when known will vary 40 depending on the compound. Typically a range of concentrations will be used. In the case of uncharacterized test compounds it may not be possible, and it is not necessary to determine the concentration of the compound.
It is desirable to include various controls e.g, positive and negative controls, in the assays. In the testing of agonist activity, negative controls can include incubating with inert compounds (e.g.
compounds known not to have agonist activity) or in the absence of added compounds. Positive controls can include incubating with compounds known to have agonist activity such as the natural ligand. As will be apparent to one of ordinary skill in the art, similar (though complementary) controls can be included in assays for antagonist activity, as well as various additional controls.
A competitive assay may also be used to screen for anagonists. The assay combines a protein 10 comprising a tltNA anticodon stem-loop recognition motif (e.g. tltNA
synthetase, ribosomal protein, or peptide of the invention), tRIVA anticodon stem-loop, and test compound under appropriate conditions for a competitive inhibition assay.
Potential agonists and antagonists include small inorganic or organic molecules, peptides, polypeptides, antibodies, a mixture of molecules, peptides, polypeptides etc., or an extract made from 15 biological materials such as bacteria, plants, fungi, or animal cells or tissues. The compound may be an endogenous physiological compound or it may be a natural or synthetic compound.
The screening assays may be used in the discovery and development of therapeutics such as antibacterial or antifungal compounds. In addition, antisense sequences to the sequence encoding the novel domain i.e. tltNA anticodon stem-loop recognition motif or binding site identified herein may be 20 used to control expression of the coding sequence and thus may be used as potential therapeutics.
The peptide of the invention can be used to model small molecules which interfere with the binding of a protein comprising a tltNA anticodon stem-loop recognition motif (e.g. tRNA synthetase or ribosomal protein), with a tRlVA anticodon stem-loop in vivo. In particular, the structure of the tRlVA anticodon stem-loop sequence recognition motif, as described herein, can be applied in 25 generating synthetic analogs and mimics of the recognition sequence.
Synthetic elements can be pieced together based upon their analogy to the structural and chemical aspects of the recognition sequence motif. Such mimics and analogs may be used in blocking or inhibiting specific aspects of protein translation and may be useful as therapeutic treatments in accordance with the methods described herein.
30 Compositions While it is possible to administer an active ingredient alone it is preferable to present it as part of a pharmaceutical composition or formulation. Therefore, the peptides, complexes, antibodies, substances, and compounds of the invention may be formulated into pharmaceutical compositions for administration to subjects in a therapeutically active amount and in a biologically compatible form 35 suitable for administration in vivo i.e. a form of the peptides etc. to be administered in which any toxic effects are outweighed by the therapeutic effects. A therapeutically active amount of a pharmaceutical composition of the invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a peptide may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage 40 regime may be adjusted to provide the optimum therapeutic response.

- IS -The peptides etc. may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the peptides etc.
may be coated in a material to protect them from the action of enzymes. The peptides etc. may also be used in combination with 5 organic substances for prolongation of their pharmacologic actions. Examples of such organic substances are non-antigenic gelatin, carboxymethylcellulose, sulfonate or phosphate ester of alginic acid, dextran, polyethylene glycol and other glycols, phytic acid, polyglutamic acid, and protamine.
The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such 10 that an effective quantity of a peptide etc. is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Phanmaceutical Sciences, Mack Publishing Company, Euston, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the peptides in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a 15 suitable pH and iso-osmotic with the physiological fluids. The peptides etc. may also be incorporated in liposomes or similar delivery vehicles.
Applications The peptides and complexes of the invention interfere with the interaction of a protein comprising a tRNA anticodon stem-loop recognition motif (e.g. tRNA synthetase or ribosomal 20 protein), and a tRNA anticodon stem-loop. The activity of a peptide or complex of the invention may be confirmed by assaying for the ability of the peptide or complex to interfere with the interaction of, for example, a tyrosyl tRNA synthetase or ribosomal S4 protein and a tRNA
anticodon stem-loop.
Computer modelling techniques known in the art may also be used to observe the interaction of a peptide of the invention, and truncations and analogs thereof with a tRNA
anticodon stem-loop (for 25 example, Homology Insight II and Discovery available from BioSym/Molecular Simulations, San Diego, California, U.S.A.). If computer modelling indicates a strong interaction, the peptide can be synthesized and tested for its ability to interfere with the interaction of a tRNA anticodon stem-loop, and a protein comprising a tRNA anticodon stem-loop recognition motif ( e.g.
tyrosyl tRNA synthetase or ribosomal S4 protein).
30 The peptides, compositions, complexes, and antibodies of the invention, and compounds and substances identified using the screening assays of the invention may be used in therapeutic applications for the treatment of living organisms including human or non-human mammalian subjects.
Alternatively, the peptides etc. may be useful as a prophylactic treatment, or in screening for compounds effective in prophylactic treatments.
35 The peptides, complexes, compositions, antibodies, and compounds and substances identified using the screening assays of the invention may be used to treat or prevent infections caused by bacteria such as species of Bacillus, E.coli, Mycobacterium, Nelicobacter, Hemophilus, Streptococcus, and Staphylococcus, and infections caused by fungi such as yeast including S.
cerevisiae, and Aspergillus. The peptides, complexes, compositions, antibodies, and compounds may also be used as 40 immunotoxins, and anti-viral agents. They may also be used as plant toxins either as an applied pesticide formulation or by incorporation into the genome of the plant. They may also be useful in vitro to arrest protein translation in biochemical assays where a precise termination of the reaction is desired.
The peptides and complexes of the invention may also be used to induce an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a peptide, complex, or composition of the invention adequate to produce antibody to protect the individual from disease (e.g. bacterial infections). An immunological response may also be induced by delivering through gene therapy a gene encoding a peptide of the invention in vivo in order to induce an immune response to protect the individual from disease. Thus, the invention contemplates a vaccine formulation which comprises a peptide or complex of the invention together with a suitable carrier.
10 The following non-limiting example is illustrative of the present invention:
EXAMPLE
The three-dimensional structure of B, stearothermophiJus tyrosyl-tRNA
synthetase (TyrRS) has been known for more than 10 years ( 1 ), however the 100 C-terminal amino acids were found disordered in the crystal structure. Deletion mutants have demonstrated that this C-terminal domain is 15 required for tRNA binding and recognition (2,3). Starting with prokaryotic TyrRS from Escherichia colt, a PSI-BLAST (4) search iteratively fords weak similarities between the C-terminal region and archeal, chloroplast and prokaryotic ribosomal S4 proteins, as well as NAM-9 the yeast mitochondrial ortholog of S4. The corresponding alignment and the motif inferred from this similarity are shown in Figure 1.
20 Ribosomal S4 protein is a multifunctional ribosomal protein associated with the 30S subunit, comprising 206 amino acids in E. colt. S4 is an ancient, if not one of the most ancient of ribosomal proteins (5). It has been demonstrated that in E. colt S4 has an autoregulatory function, binding to a pseudoknot of its own operon mRNA which limits its expression through a feedback mechanism (6). In the ribosome, S4 is required and it is the first protein involved in the folding of 16S rRNA (7).
25 Mutations of S4 proteins, specifically the ram (ribosomal ambiguity) D14 and D12 mutants in E. colt (8,9,10), the omnipotent suppresser mutant SUP46 in yeast ( 11 ), and the NAM-9 mutants of yeast( 12) affect translational accuracy. Visualization of S4 in the ribosome through 3-D
electron microscopy shows that it is superimposable with the region of the A and P sites, the entry and peptidyl-tRNA
binding sites of the ribosome ( 13,14).
30 There are several experiments elucidating the interaction between ribosome and tRNA that indicate a tRNA anticodon stem-loop interaction when tRNA is in the peptidyl (P) site. In the yeast tRNA~" , the top base pairs of the anticodon helix were shown to have an effect on ribosomal binding and activity independently of anticodon in-vivo ( I 5). Hydroxyl radical probing experiments revealed that the anticodon stem-loop is protected in the P site of 30S ribosomal subunit ( 16). Ribosomal 35 protein S4 is also implicated in the binding of the tRNA anticodon.
Ribosomal proteins S4 and S 18 were crosslinked to chemically modified anticodon nucleotides in early in-vitro experiments ( 17, 18).
Characterization of nonsense and missense suppresser tRNAs in the context of the ram S4 mutant suggested that the level of suppression might depend upon recognition of tRNA
anticodon stem-loop structure by S4 protein ( 19). S4 ram mutations also affect the kinetic off rates of the P site tRNA
40 binding (20).

-17_ The mRNA and rRNA binding region of S4 have been previously characterized. It has been shown that both functions reside within residues 47 -104 in E. coli S4 protein (21,22). Yet the most conserved region of S4 lies to the C-terminal side of this region and has not been shown to possess a specific function to date. This region (residues 97 -I32 in E. cola also comprises the motif similarity 5 between S4 and the TyrRS C-terminal region shown in Figure 1. Interestingly, no suppressor or ribosomal ambiguity mutants have ever been characterized from this most conserved portion of S4.
Mutations here have been only rarely observed probably owing to their functional importance. A more fundamental and specific binding and recognition event is the reason for the conservation of this motif.
The lack of structure of the C-terminus in the crystals of B.
stearothermophilus TyrRS has led 10 to the question as to whether the C-terminus of TyrRS has any structure at all. Similarly, the structure of the S4 protein rRNA and mRNA binding domain has been studied, and most recently the 3-D
structure of S4 has been determined (23, 24). Circular Dichroism spectra of the in E. coli S4 fragment 48 - 177 (21 ) and the B. stearothermophilus TyrRS fragment 323-419 (25) corresponding to the motif presented here are qualitatively similar in shape, supporting a shared folded structure. Sequence-15 structure threading (26) was performed to determine a structure-based alignment to further test this hypothesis. Despite difficulties in threading very small domains, the resulting threading alignment shown in Figure 2 recreated the entire motif alignment of Figure 1, and also aligned a conserved region of positively charged residues downstream of the motif.
The experimental analysis of both the 54 protein and TyrRS indicates that the individual 20 domains comprising these proteins are modular and functionally divisible.
The sequence similarity between TyrRS and S4 may be an evolutionary event in which ribosomal protein S4 was grafted onto the C-terminus of a bacterial precursor TyrRS originally more closely related to the shorter archeal tyrosyl-tRNA synthetases, which lack this C-terminus. Perhaps it is not a coincidence that the B.
subtilis genomic sequence, which encodes two tyrosyl-tRNA synthetase genes (tyrS), has one TyrRS
25 adjacent to the single ribosomal S4 gene (27) however this close spatial relationship is not found in other complete genomes. The observation of two variants of TyrRS in prokaryotes is best explained by an ancient duplication event followed by differential loss of one or the other form {28). This duplication event probably came some time after the TyrRS-S4 chimera was formed in the prokaryotic ancestor.
30 It is clear that this motif persists in the S. cerevisiae mitochondria) form of TyrRS (mtYRS).
This indicates that any such S4 insertion event would precede a symbiotic event forming mitochondria in eukaryotes, thus perhaps it is a useful evolutionary marker. In P. anserina and N. crassa mtTyrRS, the C-terminus has digressed from this sequence, accounted for by the obvious alteration of the C-terminus to accommodate the rather large group I intron splicing polypeptide, possibly a replacement 35 of the S4-like C-terminus in some mitochondria (28). Interestingly no convincing human ESTs have yet been found with similarity to a mitochondria) TyrRS. It is possible that some eukaryotes may have replaced the mtTyrRS with the cytoplasmic TyrRS.
The earlier work of Bedouelle et al.(30) demonstrates the interaction of this fragment of B.
stearothermophilus TyrRS with its cognate tRNATn. The residues from the S4-motif (namely 8368 40 and 8371 ) were amidst the 6 basic residues which, when mutated, could not complement the temperature sensitive tyrS strain of E. coli, demonstrating their requirement in the interaction with tRNATn. These include the conserved residues shown in blue in Figure 3. The model-building studies of the same group showed that the C-terminus of TyrRS will co-localize with the anticodon helix of tRNArn. The C-terminus of the eukaryotic cytoplasmic and archeal TyrRS is shorter than that of the 5 prokaryotic variants, lacking this motif. In a corresponding fashion, the tRNA binding specificity of eukaryotic tyrosyl-tRNA synthetase seems independent of the anticodon helix as the TyrRS binding affinity resides mainly in aminoacyl-stem of tRNATn (31, 32). Specificity for binding the anticodon of tRNATn in prokaryotic TyrRS may have indeed arisen by the apparent ancient addition of S4 sequence to the TyrRS C-terminus forming a chimera.
10 The comparison of ribosomal S4 protein against structures in the PDB using VAST (33) shows similarities to the Ets-fold DNA binding proteins as previously reported by the authors of the S4 structure (24). The compact substructure comprising the S4 signature motif shown in Figure 3 is not on the same side of the domain as the helix corresponding to the DNA binding helix of Ets-domain proteins. The similarity with the Ets-domain comprises this helix and the beta-sheet, but the compact 15 substructure formed by the S4 motif is not found present in any other structure in the current structure database.
All 6 of the residues identified as crucial for tRNA binding in the TyrRS C-terminus lie in this compact substructure, extended along the C-terminal side of the motif as shown as solid cartoons in Figure 3. This similarity indicates that this portion of S4 may form part of the tRNA P site binding 20 domains. A neighbor joining clustering analysis was performed using ClustalX on the motif in Figure 1 itself. Despite this being a very short sequence in the alignment, there is suffcient information to create a remarkable tree shown in Figure 4, with a nested canonical archea/eukaryote/prokaryote/chloroplast classification. Each of the mitochondria) S4 sequences which correspond to alternative mitochondria) genetic code are outgroups to this classification, with the 25 earliest branching R. americana S4 motif corresponding to the most primitive mitochondrion yet discovered (33). Since the molecular carriers of the genetic code are the cognate pool of all tRNA's, this unique clustering supports the conclusion that this is a tRNA binding motif. This nested, compact fragment within S4, indeed the most conserved region of all S4 proteins, forms a portion of the ribosomal tRNA binding P site that binds all tRNAs in the organism or organelle. This new 30 information, taken together with the mapping of the region conferring translational accuracy onto the S4 structure (24) on the opposite side of the S4 "waist" from this motif indicates that ribosomal S4 protein may comprise a significant portion of the anticodon-codon decoding active site of all ribosomes.
35 Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Below full citations are set out for the references referred to in the specification is a listing and detailed legends for the figures are provided.
The application contains sequence listings which form part of the application.

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1.
SEQUENCE LISTING
<110> Mount Sinai Hospital et al.
<120> Novel tRNA Binding Domain <130> P170PCT4 <140> PCT/CA99/00779 <141> 1999-08-24 <150> US 60/097,670 <151> 1998-08-24 <160> 48 <170> PatentIn Ver. 2.0 <210> 1 <211> 36 <212> PRT
<213> R. americana <400> 1 Leu Asp Ile Ile Ile Tyr Arg Ala Gly Phe Val Asn Ser Ile Tyr Gln Ala Arg Leu Leu Val Asn His Lys His Val Leu Val Asn Asn Lys Ile SUBSTITUTE SHEET (RULE 2B) Gln Asn Ile Ser <210> 2 <211> 36 <212> PRT
<213> M. polymorpha <400> 2 Leu Asp Val Ile Leu Val Arg Leu Asn Phe Cys Ser Thr Met Phe Gln Ala Arg Gln Leu Ile Ser His Lys Asn Ile Cys Val Asn Tyr Lys Lys Val Asn Ile Pro <210> 3 <211> 36 <212> PRT
<213> S. cerevisiae <400> 3 Leu Asp Phe Ala Leu Phe Arg Ala Met Phe Ala Ser Ser Val Arg Gln SUBSTfTUTE SHEET (RULE 26) Ala Arg Gln Phe Ile Leu His Gly Asn Val Arg Val Asn Gly Val Lys Ile Lys His Pro <210> 4 <211> 36 <212> PRT
<213> A. castellanii <400> 4 Leu Glu Asn Phe Leu Met Arg Leu Asn Leu Phe Pro Ser Ile Tyr Phe Ile Lys Lys Phe Ile Glu Tyr Gly Asn Val Phe Val Asn Asn Lys Ile Ile Asn Tyr Thr <210> 5 <211> 36 <212> PRT
<213> S. cerevisiae SUBSTITUTE SHEET (RULE 26) <400> 5 Asp Leu Ile Lys Leu Ile Cys Lys Leu Val Asn Cys Ser Val Ser Glu Ala Arg Arg Lys Leu Ser Gln Gly Ser Val Tyr Leu His His Ser Lys Ser Lys Val Asn <210>6 <211>36 <212>PRT

<213>M. genitalium <400> 6 Leu Ile Asp Tyr Leu Val Glu Thr Lys Phe Ile Lys Ser Lys Ser Glu Ala Arg Arg Leu Ile Ser Gln Lys Gly Leu Thr Ile Asn Asn Lys His Val Leu Asp Leu <210> 7 <211> 36 SUBSTITUTE SHEET (RULE 26) <212> PRT
<213> H. influenzae <400> 7 Leu Ala Thr Leu Leu Lys Glu Ala Gly Leu Val Pro Ser Thr Ser Glu Ala Ile Arg Ser Ala Gln Gln Gly Gly Val Lys Ile Asn Gly Glu Lys Val Asp Asn Val <210> 8 <211> 36 <212> PRT
<213> M.pneumoniae <400> 8 Leu Val Asp Val Ile Val Asp Leu Gly Leu Val Val Ser Arg Ser Glu Ala Arg Arg Val Ile Gln Gln Gly Gly Leu Thr Ile Asn Gln Glu Lys Val Thr Asp Val SUBSTITUTE SHEET (RULE 26) <210> 9 <211> 36 <212> PRT
<213> B. subtilis <400> 9 Met Ile Asp Leu Leu Val Lys Leu Lys Leu Leu Ser Ser Lys Ser Glu Ala Arg Arg Met Ile Gln Asn Gly Gly Val Arg Ile Asp Gly Glu Lys Val Thr Asp Val <210>10 <211>36 <212>PRT

<213>Synechocystis <400> 10 Leu Ala Tyr Leu Leu Ser Ala Ser Gly Leu Cys Pro Ser Ser Ser Glu Gly Arg Arg Gln Ile Lys Gly Gly Ala Val Arg Leu Asp Gly Asp Arg SUBSTITUTE SHEET (RULE 28) WO 00!11141 PCTICA99I00779 Leu Glu Asp Val c210> 11 <211> 36 <212> PRT
<213> T. ferrooxidans <400> 11 Leu Ser Gln Leu Leu Val Gln Val His Leu Ala Ala Ser Thr Ser Glu Ala Met Arg Lys Met Lys Glu Gly Ala Val Arg Val Asp Trp Arg Arg Val Val Asp Pro <210> 12 c211> 36 <212> PRT
<213> B. subtilis <400> 12 Leu Val Asp Val Leu Val Gln Ser Lys Leu Ser Pro Ser Lys Arg Gln SUBSTITUTE SHEET (RULE 2B) Ala Arg Glu Asp Ile Gln Asn Gly Ala Val Tyr Ile Asn Gly Glu Arg Gln Thr Glu Ile <210> 13 <211> 36 <212> PRT
<213> B. stearothermophilus <400> 13 Leu Val Glu Leu Leu Val Ser Ala Gly Ile Ser Pro Ser Lys Arg Gln Ala Arg Glu Asp Ile Gln Asn Gly Ala Ile Tyr Val Asn Gly Glu Arg Leu Gln Asp Val <210>14 <211>36 <212>PRT

<213>E.
coli <400> 14 SUBSTITUTE SHEET (RULE 26) Leu Met Gln Ala Leu Val Asp Ser Glu Leu Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro <210> 15 <211> 36 <212> PRT
<213> E. coli <400> 15 Leu Asp Asn Val Val Tyr Arg Met Gly Phe Gly Ala Thr Arg Ala Glu Ala Arg Gln Leu Val Ser His Lys Ala Ile Met Val Asn Gly Rrg Val Val Asn Ile Ala <210> 16 <211> 36 <212> PRT
SUBSTITUTE SHEET (RULE 26) <213> H. influenzae <400> 16 Leu Asp Asn Val Val Tyr Arg Met Gly Phe Ala Thr Thr Arg Ala Glu Ala Arg Gln Leu Val Ser His Lys Ala Ile Val Val Asn Gly Arg Val Val Asn Ile Pro <210> 17 <211> 36 <212> PRT
<213> B. aphidicola <400> 17 Leu Asp Asn Val Val Tyr Arg Met Gly Phe Gly Cys Thr Arg Ser Glu Ser Arg Gln Leu Ile Ser His Lys Ser Ile Lys Val Asn Asn Asn Ile Val Asn Ile Ala SUBSTITUTE SHEET (RULE 2B) <210> le <211> 36 <212> PRT
<213> M. bovis <400> 18 Leu Asp Asn Val Ile Tyr Arg Ala Gly Leu Ala Arg Thr Arg Arg Met Ala Arg Gln Leu Val Ser His Gly His Phe Asn Val Asn Gly Val His Val Asn Val Pro <210> 19 <211> 36 <212> PRT
<213> M. genitalium <400> 19 Leu Asp Asn Ile Val Tyr Arg Met Gly Phe Ala Pro Thr Arg Lys Ser Ala Arg Gln Met Val Asn His Gly His Val Ile Leu Asn Asp Gln Thr Val Asp Thr Pro SUBSTITUTE SHEET (RULE 26) <210> 20 <211> 36 <212> PRT
<213> M pneumoniae <400> 20 Leu Asp Asn Ile Val Tyr Arg Met Gly Phe Ala Pro Thr Arg Arg Ser Ala Arg Gln Leu Val Asn His Gly His Val Leu Leu Asn Asp Arg Thr Val Asp Thr Pro <210> 21 <211> 36 <212> PRT
<213> H. pylorii <400> 21 Leu Asp Asn Val Val Tyr Arg Met Gly Phe Ala Thr Thr Arg Ser Ser Ala Arg Gln Leu Val Thr His Gly His Val Leu Val Asp Gly Lys Arg SUBSTITUTE SHEET (RULE 28) Leu Asp Ile Pro <210> 22 <211> 36 <212> PRT
<213> B. sublitis <400> 22 Leu Asp Asn Val Val Tyr Lys Leu Gly Leu Ala Arg Thr Arg Arg Gln Ala Arg Gln Leu Val Asn His Gly His Ile Leu Val Asp Gly Ser Arg Val Asp Ile Pro <210> 23 <211> 36 <212> PRT
<213> B. stearothermophilus <400> 23 Leu Asp Asn Leu Val Tyr Arg Leu Gly Leu Ala Arg Thr Arg Arg Gln SUBSTITUTE SHEET (RULE 26) Ala Arg Gln Leu Val Thr His Gly His Ile Leu Val Asp Gly Ser Arg Val Asn Ile Pro <210> 24 <211> 36 <212> PRT
<213> S. oleracea <400> 24 Leu Asp Asn Ile Leu Phe Arg Leu Gly Met Ala Pro Thr Ile Pro Gly Ala Arg Gln Leu Val Asn His Arg His Ile Leu Val Asn Gly Arg Ile Val Asp Ile Pro <210> 25 <211> 36 <212> PRT
<213> O. sativa SUBSTITUTE SHEET (RULE 26) <400> 25 Leu Asp Asn Ile Leu Phe Arg Leu Gly Met Ala Ser Thr Ile Pro Glu Ala Arg Gln Leu Val Asn His Arg His Ile Leu Val Asn Gly Arg Ile Val Asp Ile Pro <210> 26 <211> 36 <212> PRT
<213> N. tabacum <400> 26 Leu Asp Asn Ile Leu Phe Arg Leu Gly Met Ala Ser Thr Ile Pro Ala Ala Arg Gln Leu Val Asn His Arg His Ile Leu Val Asn Gly Arg Ile Val Asp Ile Pro <210> 27 SUBSTITUTE SHEET (RULE 26) <211> 36 <212> PRT
<213> M. polymorpha <400> 27 Leu Asp Asn Ile Ile Phe Arg Leu Gly Met Ala Pro Thr Ile Pro Gly Ala Arg Gln Leu Val Asn His Arg His Ile Leu Ile Asn Asn Asn Thr Val Asp Ile Pro <210>28 <211>36 <212>PRT

<213>Synechocystis <400> 28 Leu Asp Asn Thr Val Phe Arg Leu Gly Met Ala Gly Thr Ile Pro Gly Ala Arg Gln Leu Val Cys His Gly His Ile Thr Val Asn Gly Gln Val Val Asp Ile Pro SUBSTITUTE SHEET (RULE 28) wo oon i ia~ Pc~r~cA99roo~~9 <210> 29 <211> 36 <212> PRT
<213> C. reinhardtii <400> 29 Leu Asp Asn Ile Val Phe Arg Leu Asn Met Ala Pro Thr Ile Pro Ala Ala Arg Gln Leu Ile Ser His Gly His Ile Arg Val Asn Asn Lys Lys Val Asn Ile Pro <210> 30 <211> 37 <212> PRT
<213> Crypt. Phi <400> 30 Leu Asp Asn Val Ile Phe Arg Leu Gly Met Ala Pro Thr Thr Ile Pro Ala Ala Arg Gln Leu Val Asn His Gly His Ile Lys Val Asn Asn Thr SUBSTITUTE SHEET (RULE 25) 1$
Arg Val Ser Ile Pro <210> 31 <211> 35 <212> PRT
<213> C. vulgaris <400> 31 Leu Asp Thr His Phe Arg Leu Gly Phe Ala Pro Thr Ile Ala Ala Ala Arg Gln Leu Ile Asn His Gly His Ile Val Val Asn Gly Arg Arg Val Asp Ile Pro <210> 32 <211> 36 <212> PRT
<213> D. melanogaster <400> 32 Leu Gln Thr Gln Val Phe Lys Leu Gly Leu Ala Lys Ser Ile His His SUBSTITUTE SHEET {RULE 28) Ala Arg Val Leu Ile Arg Gln Arg Thr Phe Val Leu Ala Ser Arg Trp Ser Thr Ile Pro <210> 33 <211> 36 <212> PRT
<213> T. brucei <400> 33 Leu Gln Thr Val Val Phe Lys His Gly Leu Ala Lys Ser Val His His Ser Arg Val Leu Ile Gln Gln Arg His Ile Ala Val Ala Lys Gln Ile Val Thr Ile Pro <210> 34 <211> 36 <212> PRT
<213> S. cerevisiae SUBSTITUTE SKEET (RULE 26) <400> 34 Leu Gln Thr Gln Val Tyr Lys Leu Gly Leu Ala Lys Ser Val His His Ala Arg Val Leu Ile Thr Gln Arg His Ile Ala Val Gly Lys Gln Ile Val Asn Ile Pro <210> 35 <211> 36 <212> PRT
<213> R. norvegicus <400> 35 Leu Gln Thr Gln Val Phe Lys Leu Gly Leu Ala Lys Ser Ile His His Ala Arg Val Leu Ile Arg Gln Arg His Ile Arg Val Leu Lys Gln Val Val Asn Ile Pro <210> 36 <211> 36 SUBSTITUTE SHEET (RULE 26) <212> PRT
<213> H. sapiens <400> 36 Leu Gln Thr Gln Val Phe Lys Leu Gly Leu Ala Lys Ser Ile His His Ala Arg Val Leu Ile Arg Gln Arg His Ile Arg Val Arg Lys Gln Val Val Asn Ile Pro <210> 37 <211> 36 <212> PRT
<213> s. pombe <400> 37 Leu Gln Thr Gln Val Phe Lys Leu Gly Leu Ala Lys Ser Ile His His Ala Arg Val Leu Ile Phe Gln Arg His Ile Arg Val Gly Lys Gln Ile Val Asn Val Pro SUBSTITUTE SHEET (RULE 28) <210> 38 <211> 36 <212> PRT
<213> P. anserina <400> 38 Leu Gln Thr Leu Val Tyr Lys Leu Gly Leu Ala Lys Ser Ile His His Ala Arg Val Leu Ile Arg Gln Arg His Ile Arg Val Gly Lys Gln Ile Val Asn Val Pro <210> 39 <211> 36 <212> PRT
<213> D. discoidium <400> 39 Leu Gln Thr Leu Val Phe Lys Asn Gly Leu Ala Lys Ser Ile His His Ala Arg Val Leu Ile Lys Gly Arg His Ile Arg Va1 Gly Lys Gln Leu SUBSTITUTE SHEET (RULE 26) Val Asn Val Pro <210> 40 <211> 36 <212> PRT
<213> N. fowleri <400> 40 Leu Gln Thr Val Val Gln Lys Leu Gly Leu Ser Lys Ser Ile His His Ala Arg Gln Leu Ile Phe Gln Arg His Ile Arg Val Gly Lys Gln Thr Val Asn Val Pro <210> 41 <211> 36 <212> PRT
<213> C. elegans <400> 41 Leu Gln Thr Gln Val Phe Lys Leu Gly Leu Ala Lys Ser Ile His His SUBSTITUTE SHEET (RULE 28) Ala Arg Ile Leu Ile Lys Gln His His Ile Arg Val Arg Arg Gln Val Val Asp Val Pro <210> 42 <211> 36 <212> PRT
<213> H. marismortui <400> 42 Leu Gln Thr Val Val Tyr Arg Lys Gly Tyr Ala Asn Thr Pro Glu Gln Ala Arg Gln Phe Ile Val His Gly His Ile Val Leu Asp Asp Ala Arg Val Thr Arg Pro <210>43 <211>36 <212>PRT

<213>S. acidocaldarius <400> 43 SUBSTITUTE SHEET (RULE 26) WO 00/11 ldl PCT/CA99/00779 Leu Gln Thr Ile Val Tyr Lys Lys Gly Leu Ala Arg Thr Ile Tyr Gln Ala Arg Gln Leu Ile Thr His Gly His Ile Ala Ile Ser Gly Arg Lys Val Thr Ser Pro <210> 44 <211> 36 <212> PRT
<213> s. solfataricus <400> 44 Leu Gln Thr Ile Val Tyr Lys Lys Gly Leu Ser Asn Thr Ile Tyr Gln 1 5 10 . 15 Ala Arg Gln Leu Ile Thr His Gly His Ile Ala Val Asn Gly Lys Arg Val Thr Ser Pro <210> 45 <211> 36 <212> PRT
SUBSTITUTE SHEET (RULE 2B) <213> M. jannaschii <400> 45 Leu Gln Thr Leu Val Phe Arg Lys Gly Leu Ala Arg Thr Pro Arg Gln Ala Arg Gln Leu Ile Val His Gly His Ile Ala Val Asn Gly Arg Val Val Thr Ala Pro <210>46 <211>63 <212>PRT

<213>Synechocystis <400> 46 Leu Ala Tyr Leu Leu Ser Ala Ser Gly Leu Cys Pro Ser Ser Ser Glu Gly Arg Arg Gln Ile Lys Gly Gly Ala Val Arg Leu Asp Gly Asp Arg Leu Glu Asp Val Asn Gln Glu Tyr Ala Asp Pro Lys Met Leu Ile Asn Lys Val Leu Gln Met Gly Lys Lys Lys Phe Ile Arg Leu Ile Ser SUBSTITUTE SHEET (RULE 26) <210> 47 <211> 66 <212> PRT
<213> B. stearothermophilus <400> 47 Leu Asp Asn Leu Val Tyr Arg Leu Gly Leu Ala Arg Thr Arg Arg Gln Ala Arg Gln Leu Val Thr Asn Gly His Ile Leu Val Asp Gly Ser Arg Val Asn Ile Pro Ser Tyr Arg Val Lys Pro Gly Gln Thr Ile Ala Val Arg Glu Lys Ser Arg Asn Leu Gln Val Ile Lys Glu Ala Leu Glu Ala Asn Asn <210> 48 <211> 85 <212> PRT
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 2B) <220>
<223> Description of Artificial Sequence: tRNA binding domain <400> 48 Met Leu Xaa Xaa Xaa Leu Ile Val Met Cys Xaa Xaa Xaa Gly Met Asn Glu His Lys Met Leu Phe Tyr Ile Xaa Xaa Ser Thr Xaa Xaa Xaa Ser Ala Ile Gly Arg Met Ile Lys Xaa Xaa Met Ile Val Ala Xaa Xaa Gly Asn Arg His Lys Xaa Ile Val Leu Phe Xaa Leu Ile Val Asn Asp Ser Arg Gly Ala Leu Xaa Xaa Xaa Leu Ile Val Ser Gln Xaa Xaa Pro Ile Leu Val Thr Ala Cys SUBSTITUTE SHEET (RULE 26)

Claims (19)

WE CLAIM:

1. A substantially pure peptide comprising the following sequence [ML]-X(3)-[LIVMC]-X(3)-[GMNEHK]-[MLFYI]-X(2)-[ST]-X(3)-[SAIG]-[RMIK]-X(2)-[MIVA]-X(2)-[GNRHK]-X-[IVLF]-X-[LIV]-[NDSRGAL]-X(3)-[LIVSQ]-X(2)-[PILVTAC]
wherein X represents any amino acid.

2. A substantially pure peptide comprising the sequence motif Y1-X(3)-Y2-X(3)-Y3-Y4-X(2)-Y5-X(3)-Y6-Y-X(2)-Y8-X(2)-Y9-X-Y10-X-Y11-Y12-X(3)-Y13-X(2)-Y14 where Y1 is methionine or leucine, preferably leucine, Y3 is leucine, isoleucine, valine, methionine, or cysteine, preferably isoleucine, leucine, or valine,Y3 is glycine, methionine, asparagine, glutamic acid, histidine, or lysine, preferably glycine, Y4 is methionine, leucine, phenylalanine, tyrosine, or isoleucine, preferably phenylalanine, leucine, methionine, or tyrosine, Y5 is serine or threonine, Y6 is serine, alanine, isoleucine, or glycine, preferably alanine, Y7 is arginine, methionine, isoleucine, or lysine, preferably arginine, Y8 is methionine, isoleucine, valine, alanine, preferably valine or isoleucine, Y9 is glycine, asparagine, arginine, histidine, or lysine, preferably lysine, glycine, or arginine, Y10 is isoleucine, valine, leucine, or phenylalanine, preferably valine or isoleucine, Y11 is leucine, isoleucine, or valine, preferably valine or isoleucine, Y12 is asparagine, aspartic acid, serine, arginine, glycine, alanine, or leucine, preferably asparagine, aspartic acid, or glycine,Y13 is leucine, isoleucine, valine, serine, or glutamine, preferably glutamine or valine, Y14 is proline, isoleucine, leucine, valine, threonine, alanine, cysteine, or serine, preferably proline or valine, and X
is any amino acid.

3. A peptide as claimed in claim 2 wherein the peptide is from 10 to 200 amino acids in length and comprises the core sequence Y1-X(3)-Y2-X(3)-Y3-Y4-X(2)-Y5-X(3)-Y6-Y-X(2)-Y8-X(2)-Y9-X-Y10-X-Y11-Y12-X(3)-Y13-X(2)-Y14.

4. A peptide as claimed in claim 2 wherein the peptide is from 30 to 75 amino acids in length and comprises the core sequence Y1-X(3)-Y2-X(3)-Y3-Y4-X(2)-Y5-X(3)-Y6-Y7-X(2)-Y8-X(2)-Y9-X-Y10-X-Y11-Y12-X(3)Y13-X(2)-Y14.

5. A peptide as claimed in claim 2 wherein the peptide is from 35 to 65 amino acids in length and comprises the core sequence Y1-X(3)Y2-X(3)-Y3-Y4-X(2)-Y5-X(3)-Y6-Y7-X(2)-Y8-X(2)-Y9-X-Y10-X-Y11-Y12-X(3)-Y13-X(2)-Y14.

6. A peptide as claimed in claim 2 that is a sequence of SEQ.ID.NOs. 1 to 47.

7. A peptide as claimed in claim 2 that binds to a tRNA anticodon stem-loop.

8. A complex comprising a peptide as claimed in claim 2 with a tRNA anticodon stem-loop.

9. An antibody specific for a peptide as claimed in claim 2 or a complex as claimed in claim 8.

10. A method for determining whether a nucleic acid comprises a tRNA anticodon stem-loop comprising the steps of contacting a nucleic acid with a peptide as claimed in claim 2 and determining whether the peptide binds to the nucleic acid, wherein the binding of the peptide to the nucleic acid indicates that the nucleic acid comprises a tRNA anticodon stem-loop.

11. A method for identifying a substance which binds to a peptide as claimed in claim 2 comprising reacting the peptide with at least one substance which potentially can bind with the peptide, under conditions which permit the formation of conjugates between the substance and peptide, and detecting binding.

12. A method as claimed in claim 1 I wherein binding is detected by assaying for conjugates, for free substance, or for non-complexed peptide.

13. A method of determining whether a test compound is an agonist or antagonist of a tRNA
synthetase-tRNA anticodon stem-loop interaction or ribosomal S4 protein-tRNA
anticodon stem-loop interaction which comprises the steps of (a) incubating the test compound with a nucleic acid comprising a tRNA anticodon stem-loop, and a peptide as claimed in claim 2; (b) determining the amount of nucleic acid bound to the peptide during the incubating step; and (c) comparing the amount of nucleic acid bound to the peptide during the incubating step to an amount of nucleic acid bound to peptide in the absence of the test compound, wherein an increase in the amount of nucleic acid bound to peptide in the presence of the test compound indicates that the test compound is an agonist of a tRNA synthetase-tRNA
anticodon stem-loop interaction or a ribosomal S4 protein-tRNA anticodon stem-loop interaction, while a decrease indicates that the test compound is an antagonist of an interaction.

14. A method for obtaining a substantially pure nucleic acid comprising a tRNA
anticodon stem-loop from a mixture of different nucleic acids comprising the steps of (a) providing a peptide as claimed in claim 2 bound to a solid support; (b) contacting the mixture of different nucleic acids with the peptide bound to the solid support whereby a nucleic acid comprising a tRNA
anticodon stem-loop is bound to the peptide; and (c) washing the solid support to remove unbound nucleic acids and eluting substantially pure nucleic acids comprising a tRNA
anticodon stem-loop from the solid support.

15. A method of interfering with the interaction of a peptide as claimed in claim 2 with a tRNA
anticodon stem-loop comprising contacting the tRNA anticodon stem-loop with the peptide.

16. A method of modulating protein synthesis comprising changing the following sequence motif in a tRNA synthetase or ribosomal protein:
[ML]-X(3)-[LIVMC]-X(3)-[GMNEHK]-[MLFYI]-X(2)-[ST]-X(3)-[SAIG]-[RMIK]-X(2)-[MIVA]-X(2)-[GNRHK]-X-[IVLF]-X-[LIV]-[NDSRGAL]-X(3)-[LIVSQ]-X(2)-[PILVTAC], wherein X is any amino acid.

17. A pharmaceutical composition for inhibiting the interaction of a tRNA, with a tRNA
synthetase or ribosomal protein comprising a peptide as claimed in claim 2 and a pharmaceutically acceptable carrier.

18. An antibacterial agent, anti-viral agent, immunotoxin, or plant toxin comprising a peptide as claimed in claim 2 or a complex as claimed in claim 3.

19. Use of a peptide as claimed in claim 2 or a complex as claimed in claim 3 in the preparation of an antibacterial agent, anti-viral agent, immunotoxin, or plant toxin.