Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-08-09
2003-10-28
Allen, Marianne P. (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C702S019000, C702S027000, C530S350000
Reexamination Certificate
active
06638908
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally pertains to the fields of molecular biology, protein crystallization, x-ray diffraction analysis, three-dimensional structural determination, structure based rational drug design and molecular modeling of ribosomes and ribosomal subunits. The present invention provides crystallization methods as well as the crystallized ribosome and ribosomal subunits. The x-ray diffraction patterns of the crystals in question are of sufficient resolution so that the three-dimensional structure of ribosomes and ribosomal subunits can be determined at atomic resolution, ligand binding sites on ribosomes and ribosomal subunits can be identified, and the interactions of ligands with ribosomes and ribosomal subunits can be modeled in specific ways.
The high resolution ribosome and ribosomal subunit maps provided by the present invention and the models prepared using such maps permit the design of ligands which can function as active agents. Thus, the present invention has applications to the design of active agents which include, but are not limited to, those that find use as antifungals, insecticides, herbicides, miticides, antibacterials, antiprotozoals and rodenticides.
BACKGROUND
Ribosomes: Structure, Function, and Composition
Ribosomes are ribonucleoproteins which are present in both prokaryotes and eukaryotes. They consist of about two-thirds RNA and one-third protein. Ribosomes are essential for protein synthesis. In the last step of the gene expression pathway, ribosomes translate the genomic information encoded in a messenger RNA into protein (Garrett, et al., 2000).
Ribosomes are complexes of two nonequivalent ribonucleoprotein subunits. The larger subunit (“large ribosomal subunits”) is about twice the size of the smaller (“small ribosomal subunits”). The small ribosomal subunit binds mRNA and mediates the interactions between mRNA and tRNA anticodons on which the fidelity of translation depends. The large ribosomal subunit catalyzes peptide bond formation—the peptidyl-transferase reaction of protein synthesis—and includes two different tRNA sites: the A site for the incoming aminoacyl-tRNA, which is to contribute its amino acid to the growing peptide chain, and the P site for peptidyl-tRNA complex, i.e. the tRNA linked to all the amino acids that have so far been added to the peptide chain. The large ribosomal subunit also includes a binding site for G-protein factors that assist in the initiation, elongation, and termination phases of protein synthesis. The large and small ribosomal subunits behave independently during the initiation phase of protein synthesis; however, they assemble into complete ribosomes when elongation is about to begin.
The molecular weight of the prokaryotic ribosome is about 2.6×10
6
. In prokaryotes, the small ribosomal subunit contains a 16S (Svedberg units) rRNA having a molecular weight of about 5.0×10
5
. The large ribosomal subunit contains a 23S rRNA having a molecular weight of about 1.0×10
6
and a SS rRNA having a molecular weight of about 4.0×10
5
. The prokaryotic small subunit contains 21 different proteins and its large subunit, 31 proteins. The large and small ribosomal subunits together make a 70S ribosome in procaryotes. Eukaryotic ribosomes are bigger than their prokaryotic counterparts. The large and small subunits together make an 80S eukaryotic ribosome. The small subunit of an eukaryotic ribosome includes a single 18S rRNA, while the large subunit includes a 5S rRNA, a 5.8S rRNA, and a 28S rRNA. The 5.8S rRNA is structurally related to the 5′ end of the prokaryotic 23S rRNA and the 28S rRNA is structurally related to the rest (Moore, 1998). Eukaryotic ribosomal proteins are qualitatively similar to the prokaryotic ribosomal proteins; however, the eukaryotic proteins are bigger and there are more of them (Moore, 1998).
Structural Conservation of the Large Ribosomal Subunit
While the chemical composition of large ribosomal subunits varies significantly from species to species, the sequences of their components provide unambiguous evidence that they are similar in three-dimensional structure, function in a similar manner, and are related evolutionarily. The evolutionary implications of the rRNA sequences data available is reviewed in the articles of Woese and others in part II of “
Ribosomal RNA. Structure, Evolution, processing and Function in Protein Biosynthesis
”, Zimmermann and Dahlberg, eds, CRC Press, Boca Raton, Fla., 1996. The article by Garret and Rodriguez-Fonseca in part IV of the same volume discusses the unusually high level of sequence conservation observed in the peptidyl transferase region of the large ribosomal subunit. Archeal species like
H. marismortui
have ribosomes that resemble those obtained from eubacterial species like
E. coli
in size and complexity. However, the proteins in their ribosomes are more closely related to the ribosomal proteins found in eukaryotes (Wool, I., Chan, Y. -L., & Gluck, A., Biochem. Cell Biol. 73, 933-947 (1995)).
Because of the high level of sequence conservation that characterizes the active site regions of ribosomes from different species, knowledge of the three-dimensional structure of a large ribosomal subunit from a single species belonging to a single kingdom, e.g. that of
H. marismortui
, will enable those skilled in the art both to understand the function of the critical regions of ribosomes from other species, regardless of kingdom. Thus it should be possible to for such an individual to produce useful models for the functionally significant regions of the ribosomes of higher organisms like humans and of the ribosomes from the bacteria that are their pathogens, and to understand how they might differ.
Determination of the Structure of Ribosomes
Much is what is known about ribosome structure derives from physical and chemical methods that produce relatively low-resolution information. Electron microscopy (EM) has contributed to the understanding of ribosome structure ever since the ribosome was discovered. In the 1970s, low resolution EM revealed the shape and quaternary organization of the ribosome. By the end of 1980s, the positions of the surface epitopes of all the proteins in the
E. coli
small subunit, as well as many in the large subunit, had been mapped using immunoelectron microscopy techniques (Oakes et al., 1986; Stoeffler et al., 1986). In the last few years, advances in single-particle cryo-EM and image reconstruction have led to three dimensional reconstructions of the
E. coli
70S ribosome and its complexes with tRNAs and elongation factors at resolutions between 15 Å and 25 Å (Stark et al., 1995; Frank et al., 1995; Stark et al., 1997a; Agrawal et al., 1996; Stark et al., 1997b). Additionally, three-dimensional, electron microscopic images of the ribosome have been produced at resolutions sufficiently high so that many of the proteins and nucleic acids that assist in protein synthesis can be visualized bound to the ribosome (Agrawal et al., 2000), and earlier this year an approximate model of the RNA structure in the large subunit was constructed to fit a 7.5 Å resolution electron microscopic map of the 50S subunit from
E. coli
as well as biochemical data (Mueller et al., 2000).
While the insights provided by electron microscopy have been useful, it has long been recognized that a full understanding of ribosome structure would derive only from X-ray crystallography. Crystallization studies of the ribosome began two decades ago by Ada Yonath and coworkers opened the possibility of using X-ray crystallography to determine the structure of the ribosome at atomic resolution. This was a challenging enterprise. Crystals of ribosomes have been especially difficult to obtain because of their huge size and their lack of internal symmetry. Moreover, since their surface is composed of highly degradable RNA and loosely held proteins, ribosomes exhibit inherent flexibility and instability. In 1979, Yonath and Wittman obtained potentially useful crystals of ribosomes and
Ban Nenad
Hansen Jeffrey
Moore Peter B.
Nissen Poul
Steitz Thomas A.
Allen Marianne P.
Mahatan C.
Testa Hurwitz & Thibeault LLP
Yale University
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