Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1996-01-26
2001-04-10
Sisson, Bradley (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S091200, C435S091210, C435S194000, C530S350000, C536S023100
Reexamination Certificate
active
06214588
ABSTRACT:
BACKGROUND OF THE INVENTION
The regulation of cellular gene expression occurs primarily at the level of transcription initiation by RNA polymerase. Regulated transcription initiation by RNA polymerase II in higher eukaryotes involves the formation of a complex with general transcription factors at promoters (Sawadogo, M. and Sentenac, A.,
Ann. Rev. Biochem
. 59:711-754 (1990). One of these factors, transcription factor IID (TFIID), contains the TATA-binding protein (TBP), which is able to bind directly to promoter DNA. The remaining components of the transcription initiation complex include RNA polymerase II and the initiation factors TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and TFIIJ. These components associate with TFIID-bound promoter DNA to form a transcription initiation complex. Sequence-specific DNA-binding proteins appear to regulate the establishment and activity of transcription initiation complexes, possibly through interactions with TFIIB and TBP and additional factors that make up TFIID.
Several high molecular weight complexes containing TBP have been identified in extracts from human and Drosophila cells (Gill, G, and Tjian, R.,
Curr. Opin. Gen. Dev
. 2:236-242 (1992); Sharp, P. A.,
Cell
68:819-821 (1992)). One of these complexes is TFIID, which contains at least eight TBP-associated factors (TAFs) (Pugh B. F., and Tjian, R. J.
Genes Dev
. 5:1935-1945 (1991)). A second complex is the RNA polymerase I promoter selectivity factor, SL1, which contains TBP and three TAFs (Comai, L., et al.,
Cell
68:965-976 (1992)). A third complex is a component of the RNA polymerase III factor TFIIIB, which consists of TBP and two TAFs (Taggart, A. K. P., et al.,
Cell
71:1015-1028 (1992)). Some of the TAFs associated with these complexes appear to function as transcriptional coactivators by providing a functional link between sequence-specific regulators and TBP (Dynlacht, B. D., et al.,
Cell
66:563-576 (1991)).
The RNA polymerase II carboxyl-terminal domain (CTD) is another component of the transcription apparatus that can bind to TBP (Usheva, A., et al.,
Cell
69:871-881 (1992)). The CTD is a highly conserved and apparently unique feature of the largest subunit of RNA polymerase Il (Young, R. A.,
Ann. Rev. Biochem
. 60:689-715 (1991)). The CTD contains 26-52 repeats, depending on the organism, of the consensus heptapeptide sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Deletion mutations that remove most or all of the CTD are lethal to cells (Nonet, M., et al.,
Cell
50:909-915 (1987)). CTD partial truncation mutations cause defects in growth and inducible gene expression in vivo and produce substantial defects in transcription initiation in vitro (Liao, S. M., et al.,
Genes Dev
. 5:2431-2440 (1991)).
An important feature of RNA polymerase II molecules recruited into the initiation complex is their association with RNA polymerase-associated proteins (RAPs) (Conaway, J. W., et al.,
J. Biol. Chem
. 266:17721-17724 (1991)). Two mammalian proteins, RAP30 and RAP74, have been identified as components of the general transcription factor TFIIF (Flores, O., et al.,
J. Biol. Chem
. 263:10812-10816 (1988)).
Despite this knowledge of the components of the RNA polymerase II transcription initiation complex, two major questions have not been addressed until now. First, how do RNA polymerase II and the general initiation factors interact with one another in vivo? For example, it is not clear whether RNA polymerase II and general factors assemble in a sequential manner on promoter DNA, or whether a large complex of these components assembles prior to association with DNA. Second, how do transcriptional regulators interact with the transcription initiation complex? Thus, we do not know whether interactions occur only between regulators and the subunit of TFIID, or whether there are additional interactions with other components of the initiation complex.
SUMMARY OF THE INVENTION
The present invention relates to RNA polymerase II holoenzyme complex. An RNA polymerase II holoenzyme complex of the present invention is a multisubunit complex comprising RNA polymerase II and one, or more, regulatory components. The regulatory components include, eukaryotic regulatory proteins, for example, yeast and mammalian SRB (Suppressor of RNA polymerase B) proteins and yeast and mammalian SWI and SNF proteins. The RNA polymerase II holoenzyme is capable of initiating transcription and is responsive to activators. Additional components associated with the RNA polymerase holoenzyme can include one, or more general transcription factors (also referred to herein as GTFs) and other components necessary and sufficient for responding to transcriptional activators. The RNA polymerase II holoenzyme described herein plays a key role in the initiation of transcription in eukaryotic cellular organisms. DNA transcription by the RNA polymerase II holoenzyme is stimulated by activator proteins, a feature not observed with purified RNA polymerase II and general transcription factors alone.
Applicants have identified and characterized eukaryotic RNA polymerase II holoenzyme complexes and their components, including those of mammalian, non-mammalian (including for example, yeasts, fungi, parasites and insects) and human origin. In one embodiment, yeast regulatory proteins, identified herein as SRB2, SRB4, SRB5, SRB6, SRB7, SRB8, SRB9, SRB10 and SRB11, which act as positive and negative regulators of the activity of RNA polymerase II are described. Encompassed by this invention are yeast SRB proteins SRB2, SRB4, SRB5, SRB6, SRB7, SRB8, SRB9, SRB10 and SRB11, the amino acids encoding these SRB proteins, and variants or derivatives (e.g., mutant SRB proteins) thereof, and antibodies reactive with the SRB proteins. The SRB proteins comprise the SRB complex which is tightly associated with the RNA polymerase II carboxy terminal domain, or CTD.
Also described herein is the cloning and sequencing of the first human SRB and the purification and characterization of a mammalian RNA polymerase II holoenzyme. hSRB7 is 35% identical to ySRB7, complements a ySRB7 deletion, and, like its yeast counterpart, binds to the carboxyl terminal domain of RNA polymerase II. hSRB7 is part of a mammalian holoenzyme complex, and results described herein show that this mammalian holoenzyme complex supports activated transcription.
As further described herein, the RNA polymerase II holoenzyme of the present invention includes additional regulatory components, including global gene regulators comprising SWI and SNF gene products. The SWI and SNF gene products are collectively referred to herein as SWI/SNF proteins. The SWI/SNF proteins, or polypeptides, play a key role in the regulation of gene expression. The regulatory function of gene expression of the SWI/SNF proteins includes chromatin remodeling. More specifically, the SWI/SNF proteins provide the RNA polymerase II the holoenzyme capacity to disrupt nucleosomal DNA and, thus, facilitate stable binding of various components of the transcription initiation complex at specific promoters. The SWI/SNF proteins encompassed by the present invention form a multisubunit complex with the SRB proteins, referred to herein as the SRB/SWI/SNF complex. The SRB/SWI/SNF complex associates with the RNA polymerase II CTD. Encompassed by the present invention are the SWI/SNF proteins which comprise the SRB/SWI/SNF complex.
Also encompassed by this invention are the DNA sequences encoding the eukaryotic, e.g., yeast and mammalian, SRB proteins and novel SWI/SNF proteins, the complementary strands of these DNA sequences, and allelic variations thereof, and nucleic acid probes that are sufficiently complementary to a SRB or SWI/SNF DNA sequence that they selectively hybridize to that SRB or SWI/SNF DNA sequence.
This invention further relates to methods of modifying gene transcription by substances that bind to, or interact with, SRB proteins or SWI/SNF proteins; the SRB genes and SWI/SNF genes encoding the proteins, or the SRB or SWI/SNF mRNAs. Such substances can either prevent, or enhance, the formation of t
Chao David M.
Koleske Anthony J.
Thompson Craig M.
Young Richard A.
Darby & Darby
Sisson Bradley
Stole Einar
Whitehead Institute Biomedical Research
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