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  • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
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  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to new means for detecting and analyzing interactions between proteins.
• Protein / protein interactions are fundamental cellular mechanisms which are involved in the formation of multimeric complexes responsible for functions such as transcription, translation, as well as signal transmission, response to pathogens, etc.
• This process is based on the co-expression in the same yeast cell, of the following genes:
• a reporter gene expressing a detectable protein, the level of expression of which depends on transcriptional activation by a polypeptide domain; and - two chimeric genes, coding for two hybrid proteins comprising respectively the sequences of the two proteins whose interaction is to be detected: one further comprises a transcriptional activation domain regulating the expression of the reporter gene, and the other further comprises a DNA binding domain, which recognizes a binding site located on the reporter gene in the host cell.
• GAL4 activates the transcription by RNA polymerase II (PolII) of genes coding for enzymes involved in the metabolism of galactose.
• This protein has 2 functionally independent and physically separable domains: a DNA binding domain, represented by the N-terminal domain (amino acids 1-147) which binds to specific DNA sequences (UASQ, for Upstream Activating Sequence for Galactose), and a transcriptional activation domain represented by the C-terminal domain (amino acids 768-881), which activates transcription by PolII.
• GAL4 domain (1-147) fused to a first test protein
• the other contains the GAL4 domain (768-881) fused to a second test protein. If the 2 test proteins are able to interact, they bring the two domains together of GAL4, and trigger the transcription of the reporter gene (for example the lacZ gene coding for the ⁇ -galactosidase of E. coli.).
• the reporter gene for example the lacZ gene coding for the ⁇ -galactosidase of E. coli.
• the protein whose partners are sought is fused to the GAL4 binding domain (1-147) and it is tested against a bank of proteins fused to the GAL4 activator domain (768-881).
• FIELDS and SONG Numerous improvements have been made to the technique initially used by FIELDS and SONG: for example, a cloning system in ⁇ phages which can subsequently be converted into plasmids by recombination of lox sites has been developed; a second reporter gene, consisting of an auxotrophy marker, the HIS3 gene (involved in the metabolism of histidine), was used in combination with the lacZ gene, in order to more effectively eliminate false positives [DURFEE and al., Genes & Development, 7, 555-569, (1993)].
• a derived technique makes it possible to detect proteins which bind to DNA fragments (for example promoters or fragments involved in DNA replication).
• DNA fragments for example promoters or fragments involved in DNA replication.
• This library is introduced into cells containing a reporter gene (HIS3 or lacZ) placed downstream of the DNA fragment studied [WANG and REED, NATURE, 364, 121-126, (1993); LI and HERSKOWITZ, SCIENCE, 262, 1870-1874, (1993)].
• the inventors have undertaken the development of a system which does not have this limitation. To this end, they had the idea of looking for a method based on the use of the PolIII system. However, to achieve a double hybrid system based on the use of polymerase III, it it was necessary on the one hand to identify transcription factors of the PolIII system which can be used for this purpose, and on the other hand to have suitable reporter genes.
• the PolIII system transcribes "household" genes, the products of which (tRNA, 5S rRNA, U6 RNA, etc.) are necessary for the basic functions (translation, splicing, etc.) of any active cell. The functioning of the PolIII system requires the presence of different transcription factors, ensuring correct positioning of the polymerase III.
• the transcription of tRNA genes first involves a factor called TFIIIC or ⁇ which binds to intragenic sequences called block A and block B.
• the binding of ⁇ to block B involves the subunit ⁇ l38 and its attachment to block A, the ⁇ 95 subunit.
• ⁇ then allows the recruitment of another factor, called TFIIIB, near the site of initiation of transcription.
• TFIIIB allows the recruitment and positioning of polymerase III on the gene.
• the PolIII promoter comprises an intragenic A block, a B block downstream of the gene transcription termination signal, and a TATA box in position -30.
• a succession of steps analogous to that described for the tRNA genes makes it possible to initiate the transcription of SNR6. These steps are shown diagrammatically in Figure 1.
• the sub-unit ⁇ 138 of the factor ⁇ is fixed first on block B (1), then the sub-unit ⁇ 95 on block A (2).
• the fixation of ⁇ makes it possible to recruit TFIIIB through the subunit ⁇ l31 (3).
• the precise role of the other sub-units ( ⁇ 50, ⁇ 60 and ⁇ 91) has not yet been established.
• TFIIIB which is composed of the subunits of 70 kD (70), 90 kD (90) and TBP (TATA-binding protein, protein binding to the TATA box) is positioned upstream of SNR6 (4) and allows then recruiting the PolIII polymerase (5).
• the binding of ⁇ 138 to block B of SNR6 therefore constitutes one of the first steps in the transcriptional activation of this gene.
• a mutation in block B (deletion of the bases +238 and +239 relative to the site of initiation of transcription of SNR6) which abolishes this link prevents the transcription of the gene SNR6 [BROW and GUTHRIE, Gene & Developement 4, 1345-1356 (1990) ; BURNOL et al., Nature, 362, 475-477, (1993)].
• the inventors have found that the transcription of the mutated SNR6 gene, via the fusion protein GAL4- (1-147) - ⁇ l38 could provide enough transcripts to allow the cells to grow without the presence of the wild type SNR6 gene. .
• the inventors have introduced plasmids comprising the SNR6 gene without block B and with UASQ sequences, as well as plasmids comprising the construction GAL4- (1-147) - ⁇ l38, in the yMCM strain yMCM616.
• yMCM616 is a derivative of the YPH500 ⁇ strain
• the transformants of yMCM616 comprising the construction GAL4- (1-147) - ⁇ l38 alone, or else the construction GAL4- (1-147) - ⁇ l38 and SNR6 genes without block B and without UASQ sequences do not grow in the presence 5-fluoro-orotic acid (5-FOA).
• the yMCM616 transformants comprising the construction GAL4- (1-147) - ⁇ l38 and the SNR6 gene without block B and with UASQ sequences, are capable of growing in the presence of 5- acid. fluoro-orotic, that is to say to lose the plasmid URA3 carrying the wild SNR6 gene, which shows that the presence of the wild type SNR6 gene is not necessary for the cell viability of these transformants.
• the inventors have thus found that the presence of the three types of plasmids, (namely a plasmid carrying the construction X138-PRP21, a plasmid carrying the construction GAL4- (1-147) -PRP9 or GAL4- (1- 147) -PRP 11, and a plasmid carrying the SNR6 gene without block B and with UASQ sequences) is necessary to allow the transformants to grow in the presence of 5-fluoro-orotic acid.
• no cell growth is observed when one of the plasmids GAL4- (1-147) -PRP9 / 11 or ⁇ l38-PRP21 is missing or when the SNR6 genes devoid of block B are also devoid of the UASQ sequences.
• the inventors obtained chimeric proteins resulting, one from the fusion of GAL4- (1-147) with a protein member of an interaction couple, and the other from the fusion of ⁇ l38 with the other member of said couple. interaction couple, and found that the interaction between these two fusion proteins reproduced the activity of the transcription factor GAL4- (1-147) - ⁇ l38.
• the transcription system thus obtained is shown diagrammatically in Figure 3.
• the efficiency of transcription by this system makes it possible to obtain enough transcripts to ensure cell viability, and therefore to practice a selection test based on cell viability.
• the subject of the present invention is a chimeric protein resulting from the fusion of a polypeptide constituting a transcription activator PolIII, with a polypeptide constituting a member of a protein / protein interaction pair.
• the polypeptide constituting an activator of the transcription by the polymerase PolIII is a subunit of a transcription factor PolIII or a subunit of the polymerase PolIII itself, or represents at least part of one of these subunits.
• the polypeptide constituting an activator of the transcription by the polymerase PolIII is chosen from the group consisting of the subunits of the factor TFIIIC ( ⁇ 138, X131, etc.), and the polypeptides representing a part of said subunits.
• the present invention also relates to a gene coding for a chimeric protein as defined above, as well as a recombinant vector carrying said gene.
• the present invention also relates to a pair of chimeric proteins comprising a first chimeric protein as defined above, and a second chimeric protein resulting from the fusion of a polypeptide capable of binding to a specific DNA sequence, with a polypeptide constituting the other member of the protein / protein interaction couple mentioned above.
• the present invention also relates to a method for detecting interactions between two proteins, which method is characterized in that the following DNA sequences are introduced into the same host cell:
• the reporter gene transcribed by polymerase III is the SNR6 gene.
• PolIII genes can also be used as reporter genes, provided that their expression leads to the appearance of a phenotype observable.
• reporter genes may for example be tRNA genes, and in particular, genes for suppressing translation termination codons.
• This reporter gene (s) can be found on centromeric or multicopy plasmids, or be integrated into the chromosomes of the host cell.
• the binding site recognized by a polypeptide sequence capable of binding to DNA is a UASQ sequence, and the polypeptide which recognizes said binding site is the domain 1-147 of GAL4.
• polypeptide sequence-binding site associations can also be used, such as for example the LexA binding domain or that of the human estrogen receptor, cited above; these polypeptides are used in association with their respective DNA target sequences (operators LexA and EREs, elements of response to the estrogen receptor).
• the polypeptide constituting an activator of transcription by the polymerase PolIII is the ⁇ 138 subunit of factor TFIIIC.
• the host cell used is preferably a yeast cell (Saccharomyces cerevisiae, Schizosaccharomyces pombe).
• the yMCM616 strain is identical to the FTY115 strain described by MARSOLIER et al. [Genes & Development, 9, 410-422, (1995)], with the exception that it does not include the plasmid pRS314-U6, but the centromeric plasmid URA3 pRS316-U6, carrying the region of the SNR6 gene which extends between bases -140 to + 314 relative to the starting site of transcription.
• the plasmid pRS316-U6 was obtained from the original plasmid URA3 pRS316, which was described by. SIKORSKI and HIETER, [Genetics, 122, 19-27, (1989)].
• the region (-140, - +314) of SNR6 was cloned between the Kpnl and Sacl sites of pR316 to give pR316-U6.
• FIG. 4 This construction is shown diagrammatically in FIG. 4 (CEN: centrometric sequences of yeast; URA3: gene coding for orotidine-5 ′ -phosphate decarboxylase which is involved in the biosynthesis of uracil).
• the genotype of, the strain yMCM616 is: ⁇ , ura3-52, lys2-801 a bre , ade2-101 ocher , trpl- ⁇ 63, his3- ⁇ 200, Ieu2- ⁇ l. Furthermore, the wild-type chromosomal SNR6 gene of the initial strain has been replaced by a mutant SNR6 gene whose block B has been inactivated by the deletion of bases 238-239 (from the transcription initiation site) [MARSOLIER and al., Genes & Development, vol. 9, 410-422, (1995)].
• an SNR6 gene without block B and with UAS Q sequences similar to the "B block-UAS Q template" construction described by MARSOLIER et al., [Proc Natl. Acad. Sci. USA, 91, 11938-11942, (1994)] apart from the fact that it lacks the insertion of 24 bp at position +73, has been cloned between the Apal and SacI sites of the centromeric plasmid LEU2, pRS315 [SIKORSKI and HIETER, Genetics, 122, 19-27, (1989)] This construction is shown diagrammatically in FIG. 5 (CEN: centrometric sequences of yeast; LEU2: gene coding for ⁇ -isopropylmalate dehydrogenase involved in the synthesis of leucine).
• Plasmids respectively designated GAL4- (1-147) -PRP9, and GAL4- (1-147) -PRP11, and derivatives of pMA424 have been previously described [LEGRAIN et al. , Genes & Development, 7, 1390-1399, (1993); LEGRAIN and CHAPON, Science, 262, 108-110, (1993)]. These plasmids contain a construct comprising the entire sequence coding for PRP9 or for PRP11, fused to the 147 N-terminal amino acids of GAL4. These constructions are shown diagrammatically in FIG.
• ADH1-PRO promoter region for the transcription of the yeast gene ADH1 coding for an alcohol dehydrogenase
• ADH1-TER region which terminates the transcription of the ADH1 gene
• - 2 ⁇ autonomous replication sequences derived yeast plasmid 2 ⁇
• HIS3 gene coding for imidazoleglycerol-phosphate dehydrogenase involved in the synthesis of histidine
• the X138-PRP21 construct constituted by the entire sequence of ⁇ 138 [LEFEBVRE et al. Proceedings of the National Academy of Science USA, 89, 10512-10514, (1992)] fused to the coding region of PRP21, was obtained as follows:
• pOL101 The sequence of ⁇ 138 was cloned from the plasmid called pOL101 (supplied by Olivier LEFEBVRE, CEA-Saclay, Gif-sur-Yvette).
• pOL101 was obtained as follows: the plasmid pOL45 described by LEFEBVRE et al. , [Proceedings of the National Academy of Sciences USA, 89, 10512-10516 (1992)] which contained the sequence of tl38 had been modified to eliminate the intron of the gene (LEFEBVRE et al., 1992, already cited).
• This plasmid pOL45 modified was later mutagenized by site-directed mutagenesis [KUNKEL et al., Methods in Enzymology, 154, 367-382, (1987)]: BamHI sites were introduced, one, just in front of the initiation codon of ⁇ 138, the other, just in front of the termination codons, to give pOL101.
• the BamHI fragment of pOL101 comprising the entire coding sequence of ⁇ 138 was then cloned into the vector pBluescript SK (BSSK, Stratagene) so that the KpnI site of BSSK is located at the 5 ′ end of ⁇ 138.
• the resulting plasmid is named BSSK-X138.
• pPL247 The PRP21 sequence was cloned from the plasmid called pPL247 (this plasmid was supplied by Pierre LEGRAIN, Institut Pasteur, Paris). Briefly, pPL247 was obtained as follows: A DNA fragment was derived by PCR from the PRP21 gene. This fragment comprises the coding sequence of PRP21 framed in 5 ′ by a BamHI site introduced in -8 relative to the initiation codon, and in 3 ′ by an EcoRI site, followed by a BamHI site introduced 26 bp after the codon of termination. This BamHI digested DNA fragment was cloned into the BamHI site of pMA424
• Plasmid pYcDE-2 was provided by BD HALL, University of Washington, Seattle, USA.
• This plasmid is derived from the plasmid pMAC561 [McKNIGHT and McCONAUGHY Proc.Natl.Acad.Sci USA, 80, 4412-4416 (1983)] by deletion of a SphI fragment comprising the upstream part of the promoter of the ADH1 gene, which is thus reduced to an EcoRI-SphI fragment, which makes it truly constitutive.
• the construction resulting from the insertion of the chimeric fragment X138-PRP21, into the EcoRI site of the plasmid pYcDE-2 is shown diagrammatically in FIG. 7
• ADH1-PRO promoter region for the transcription of the yeast gene ADH2
• CYCl-TER terminator region for the transcription of the yeast gene CYCl coding for iso-1-cytochrome c
• 2 ⁇ autonomous replication sequences derived from the plasmid yeast 2 ⁇
• TRP1 gene coding for an enzyme involved in the synthesis of tryptophan
• transforming clones are selected on minimum media devoid of uracil, leucine, histidine or tryptophan (depending on the marker genes carried by the plasmids) and are then spread on complete medium in the presence of 5-fluoro-orotic acid .
• plasmids namely a plasmid carrying X138-PRP21, a plasmid carrying GAL4- (1-147) -PRP9 or 11, and a plasmid carrying the gene SNR6 lacking block B and comprising UAS sequences Q ) is necessary to allow the transformants to grow in the presence of 5-fluoro-orotic acid. No cell growth is observed when one of the plasmids GAL4- (1-147) -PRP9 / 11 or X138-PRP21 is missing, or when the SNR6 genes lacking block B are also lacking the UASQ sequences.
• the transformants containing the constructs GAL4- (1-147) -PRP9 / 11 and X138-PRP21 were subcultured several times on 5-FOA medium, and have this fact lost the plasmid URA3 containing the wild SNR6 gene.
• Their SNR6 transcripts therefore come exclusively from the transcription of the SNR6 gene without block B but with the UAS Q sequences via the 2 hybrids.
• SNR6 transcripts produced were quantified by RNA transfer, as described previously [MARSOLIER et al., Proc. Natl. Acad. Sci. USA, 91, 11938-11942, (1994)], using the SNR31 gene transcript as an internal control.
• Table I below represents the percentage of SNR6 transcripts produced relative to the yMCM616 strain carrying only the wild type SNR6 gene on the plasmid pRS316, which represents 100%.
• GAL4- (1-147) and where these two domains are therefore strongly fixed by covalent bond, a level of expression is obtained (which is arbitrarily fixed at 100%), more or less comparable to the level obtained with the activators and wild genes (190% for PolII with lacZ and 110% for PolIII with SNR6).

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