Translation driver system and methods for use thereof

Details Translation driver system and methods for use thereof Translation driver system and methods for use thereof

1999-03-08

2003-08-26

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252300, C435S325000, C536S023100, C536S023400

Reexamination Certificate

active

06610508

ABSTRACT:

1. INTRODUCTION
The present method relates to the translational activation of genes using the ribosome recruitment protein, eIF4G or an eIF4G-like protein. The invention relates to the translation of RNA molecules containing heterologous protein-binding sites, which RNA molecules encode one, two, three or more proteins. The invention provides products and methods for the identification of RNA-binding proteins. The invention further provides a system by which protein—protein interactions and inhibitors or enhancers of these interactions may be identified. Further, the invention provides products and methods to provide a cell with one or more therapeutic proteins. The invention provides products and methods for controlling the levels of translation of such proteins. The invention provides products and methods to control the translation and stoichiometry of multiple subunit proteins. The invention provides products for and methods of screening for proteins which interact with an RNA binding site, and methods for identifying RNA binding sites.
2. BACKGROUND OF THE INVENTION
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
2.1. PROTEIN—PROTEIN INTERACTIONS BY TRANSCRIPTIONAL SYSTEMS
Proteins and protein—protein interactions play a central role in the various essential biochemical processes. For example, these interactions are evident in the interaction of hormones with their respective receptors, in the intracellular and extracellular signaling events mediated by proteins, in enzyme substrate interactions, in intracellular protein trafficking, in the formation of complex structures like ribosomes, viral coat proteins, and filaments, and in antigen-antibody interactions. These interactions are usually facilitated by the interaction of small regions within the proteins that can fold independently of the rest of the protein. These independent units are called protein domains. Abnormal or disease states can be the direct result of aberrant protein—protein interactions. For example, oncoproteins can cause cancer by interacting with and activating proteins responsible for cell division. Protein-protein interactions are also central to the mechanism of a virus recognizing its receptor on the cell surface as a prelude to infection protein—protein interactions direct signal transduction cascades that result in a biological response. Identification of domains that interact with each other not only leads to a broader understanding of protein—protein interactions, but also aids in the design of inhibitors of these interactions.
Protein-protein interactions have been studied by both biochemical and genetic methods. The biochemical methods are laborious and slow, often involving painstaking isolation, purification, sequencing and further biochemical characterization of the proteins being tested for interaction. As an alternative to the biochemical approaches, genetic approaches to detect protein—protein interactions have gained in popularity as these methods allow the rapid detection of the domains involved in protein—protein interactions.
An example of a genetic system to detect protein—protein interactions is the “Two-Hybrid” system to detect protein—protein interactions in the yeast
Saccharomyces cerevisiae
(Fields and Song, 1989, Nature 340:245-246; U.S. Pat. No. 5,283,173 by Fields and Song). This assay utilizes the reconstitution of a transcriptional activator like GAL4 (Johnston, 1987, Microbiol. Rev. 51:458-476) through the interaction of two protein domains that have been fused to the two functional units of the transcriptional activator: the DNA-binding domain and the activation domain. This is possible due to the bipartite nature of certain transcription factors like GAL4. Being characterized as bipartite signifies that the DNA-binding and activation functions reside in separate domains and can function in trans (Keegan et al., 1986, Science 231:699-704). The reconstitution of the transcriptional activator is monitored by the activation of a reporter gene such as the lacZ gene that is under the influence of a promoter that contains a binding site (Upstream Activating Sequence or UAS) for the DNA-binding domain of the transcriptional activator. This method is most commonly used either to detect an interaction between two known proteins (Fields and Song, 1989, Nature 340:245-246) or to identify interacting proteins from a population that would bind to a known protein (Durfee et al., 1993, Genes Dev. 7:555-569; Gyuris et al., 1993, Cell 75:791-803; Harper et al., 1993, Cell 75:805-816; Vojtek et al., 1993, Cell 74:205-214).
Another system that is similar to the Two-Hybrid system is the “Interaction-Trap system” devised by Brent and colleagues (Gyuris et al., 1993, Cell 75:791-803). This system is similar to the Two-Hybrid system except that it uses a LEU2 reporter gene and a lacZ reporter gene. Thus protein—protein interactions also lead to the reconstitution of the transcriptional activator system and allows cells to grow in media lacking leucine and enable them to express &bgr;-galactosidase. The DNA-binding domain used in this system is the LexA DNA-binding domain, while the activator sequence is obtained from the B42 transcriptional activation domain (Ma and Ptashne, 1987, Cell 51:113-119). The promoters of the reporter genes contain LexA binding sequences and hence will be activated by the reconstitution of the transcriptional activator. Another feature of this system is that the gene encoding the DNA-binding domain fusion protein is under the influence of an inducible GAL promoter so that confirmatory tests can be performed under inducing and non-inducing conditions.
Still other versions of the two-hybrid approach exist, for example, a “Contingent Replication Assay” has been reported (Nallur et al., 1993, Nucleic Acids Res. 21:3867-3873; Vasavada et al., 1991, Proc. Natl. Acad. Sci. USA 88:10686-10690). In this case, the reconstitution of the transcription factor in mammalian cells due to the interaction of the two fusion proteins leads to the activation of transcription of the SV40 T antigen. This antigen allows the replication of the activation domain fusion plasmids. Another modification of the two-hybrid approach using mammalian cells is the “Karyoplasmic Interaction Selection Strategy” that also uses the reconstitution of a transcriptional activator (Fearon et al., 1992, Proc. Natl. Acad. Sci. USA 89:7958-7962). Reporter genes used in this case have included the gene encoding the bacterial chloramphenicol acetyl transferase, the gene for cell-surface antigen CD4, and the gene encoding resistance to Hygromycin B. In both of the mammalian systems, the transcription factor that is reconstituted is a hybrid transcriptional activator in which the DNA-binding domain is from GAL4 and the activation domain is from VP16.
Recently, a transcriptional activation system has been described to isolate and catalog possible protein—protein interactions within a population, and allow the comparison of such interactions between two populations (see PCT Publication WO 97/47763 published Dec. 18, 1997).
However, all of the assays mentioned above utilize a transcriptional activation system which examines the interaction of DNA binding proteins with DNA of a reporter gene. Additionally, the transcriptional systems require that proteins being assayed be driven into the nucleus. Accordingly, there is a need in the art for a system which allows for detecting protein—protein interactions and inhibitors or enhancers of such interactions in the cytoplasmic compartment of a cell. The present invention provides such a system.
Additionally, none of the systems described above provide a means by which a protein important for the translational-activation of a gene may be identified. Nor do any of the methods described above provide a method for activating the translation of a gene-of-interest. The present invention provides such methods and compositions as well as therapeutic, diagnostic, and analytical uses of such methods and compositio

Affiliated with

Also associated with

Rating

Translation driver system and methods for use thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Translation driver system and methods for use thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Translation driver system and methods for use thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3096832