Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-13
2001-10-16
Budens, Robert D. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S440000, C436S504000, C536S023100, C536S024100
Reexamination Certificate
active
06303299
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for studying translation initiation and for identifying potential inhibitors of translation initiation. More specifically, it relates to methods in which proteins are expressed in the presence of misacylated suppressor tRNAs.
2. Discussion of the Background
Translation initiation is a key regulatory step in the synthesis of numerous proteins in eukaryotic cells. Different forms of the same protein sometimes with absolutely distinct functions can be produced as a result of alternative initiation of translation (Bullock, et al (1997)
J. Exp. Med
., 186, 1051-1058; Prats, et al. (1992)
Mol. Cell. Biol
., 12, 4796-4805; Delmas, et al. (1992)
Proc. Natl. Acad. Sci. USA
, 89, 4226-4230 and Packham, et al. (1997)
Biochem. J
., 328, 807-813.). Translation reinitiation is involved in regulation of viral life cycle for human immunodefficiency virus type I (Schwartz, et al., (1990)
J. Virol
., 64, 5448-5456; Schwartz, et al., (1992)
Mol. Cell. Biol
., 12, 207-219 and Luukkonen, et al., (1995)
J. Virol
., 69, 4086-4094.).
The current model of translation initiation in eukaryotes postulates that protein synthesis starts at the AUG codon nearest to the 5′-end of the mRNA (Kozak, M. (1987)
Nucleic Acids Res
., 15, 8125-8148; Kozak, M. (1989),
J. Cell Biol
, 108, 229-241; Kozak, M. (1992),
Crit. Rev. Biochem. Mol. Biol
., 27, 385-402; Kozak, M. (1986),
Cell
, 44, 283-292; Kozak, M. (1987)
J. Mol. Biol
., 196, 947-950 and Kozak, M. (1989)
Mol. Cell. Biol
., 9, 5073-5080.). In some cases, reinitiation of translation can occur at the next AUG codon after termination of protein synthesis (Kozak, M. (1987) Mol. Cell. Biol., 7, 3438-3445; Hinnebusch, A. G. (1990)
Trends Biochem. Sci
., 15, 148-152 and Kozak, M. (1995)
Proc. Nati. Acad. Sci. USA
, 92, 2662-2666). Another process, called ‘leaky scanning’, takes place when the 40S ribosomal subunit bypasses the first AUG codon because of a non-optimal context. In this case the ribosomes will start synthesis at the second AUG codon (Kozak, M. (1992)
Crit. Rev. Biochem. Mol. Biol
., 27, 385-402; Kozak, M. (1986)
Cell
, 44, 283-292; Kozak, M. (1987)
J. Mol. Biol
., 196, 947-950; Kozak, M. (1989)
Mol. Cell. Biol
., 9, 5073-5080 and Kozak, M. (1995)
Proc. Natl. Acad. Sci. USA
, 92, 2662-2666).
In spite of the fact that the above-described translation initiation mechanism was shown to predominate for norm eukaryotic mRNAs, some viruses, such as hepatitis C virus (HCV), Moloney murine leukemia virus (MoMuLV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and poliovirus, use an unusual internal translation initiation process (Kolupaeva, et al. (1996)
RNA
, 2, 1199-1212; Jang, et al. (1988)
J. Virol
., 62, 2636-2643; Pellitier, et al. (1988)
Nature
, 334, 320-335; Kuhn, et al. (1990),
J. Virol
, 64, 4625-4631; Tsukiyama-Kohara, et al. (1992)
J. Virol
., 66, 1476-1483 and Vagner, et al. (1995)
J. Biol. Chem
., 270, 20376-20383). The same mechanism was recently found for translation of human proto-oncogene c-myc (Nanbru, et al. (1997)
J. Boil. Chem
., 2272, 32061-32066). In this case, protein synthesis starts far from the 5′-end (more than 400 nucleotides).
A powerful technique of incorporation of synthetic amino acid into a protein at predetermined positions was developed within the last two decades. The method involves incorporation of a stop codon at the position of interest in the protein gene and in vitro expression of the gene in the presence of misacylated suppressor tRNA, the latter of which will allow to readthrough of the stop codon (Hecht, et al. (1978)
J. Biol. Chem
., 253, 4517-4520; Heckler, et al. (1983),
J. Biol. Chem
., 258, 4492-4495; Heckler, et al. (1984),
Biochemistry
, 23, 1468-1473; Baldini, et al. (1988)
Biochemistry
, 27, 7951-7959; Bain, et al. (1989)
J Am. Chem. Soc
., 111, 8013-8014; Roesser, et al. (1989)
Biochemistry
, 28, 5185-5195; Robertson, et al. (1989)
Nucleic Acids Res
., 17, 9649-9660; Robertson, et al. (1991)
J Am. Chem. Soc
., 113, 2722-2729; Hecht, S. M. (1992)
Acc. Chem. Res
., 25, 545-552; Lodder, et al. (1997)
J. Org. Chem
., 62, 778-779; Noren, et al. (1989)
Science
, 244, 182-188; Bain, et al. (1991)
Tetrahedron
, 47, 2389-2400 and Mamaev., et al. (1996)
J. Am. Chem. Soc
., 118, 7243-7244). Numerous modified proteins and peptides have been successfully synthesized and studied using this strategy (Noren, et al. (1989)
Science
, 244, 182-188; Bain, et al. (1991)
Tetrahedron
, 47, 2389-2400; Mamaev., et al. (1996)
J. Am. Chem. Soc
., 118, 7243-7244; Ellman, et al. (1992)
Science
, 255, 197-200; Chung, et al. (1993)
Science
, 259, 806-809; Mendel, et al. (1993)
J Am. Chem. Soc
., 115, 4359-43360 and Thorson, et al. (1995)
J Am. Chem. Soc
., 117, 1157-1160). One recent achievement was exploring translation initiation mechanism using an in Vitro suppression method (Karginov, et al. (1997)
Nucleic Acids Res
., 25, 3912-3916). It was shown that different types of translation initiation (i.e. reinitiation, ‘leaky scannings’) can be regulated and distinguished by changing of concentration of the misacylated tRNA.
In view of the importance of understanding translation initiation for the development of inhibitors and/or modulators thereof, it is clear that there exists a need in the art for a method for studying translation initiation and for identifying inhibitors of translation initiation.
SUMMARY OF THE INVENTION
Accordingly, one object of this invention is to provide a novel method for analyzing a translation initiation mechanism by:
a expressing proteins encoded by a nucleic acid in the presence of a misacylated suppressor tRNA;
b. analyzing relative amounts of the proteins produced in (a); and
c. comparing the amount of each protein analyzed into (b) to determine the translation initiation site utilized for each protein.
Another object of the invention is to provide a method for identifying inhibitors of translation initiation by:
a. performing the above-noted method in the presence and absence of a potential inhibitor, on a nucleic acid from which translation initiation is to be inhibited;
b. comparing the amount of proteins produced in the presence and absence of the potential inhibitor; and
c. correlating a decrease in proteins produced in the presence of the potential inhibitor with the ability of the potential inhibitor to inhibit the translation of the proteins.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.
REFERENCES:
Roesser et al.,Biochemistry28:5185-5195, 1989.*
Heckler et al.,Biochemistry27:7254-7262, 1988.
Hecht Sidney M.
Karginov Andrei V.
Karginov Vladimir
Breen John P.
Budens Robert D.
University of Virginia Patent Foundation
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