Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
Problem Outline
Can we prevent parasites from using the host transcription machinery? Analysis
of transcription, replication, recombination and translation, in parasites, are still (1993)
virtually virgin fields. Biochemical approaches may be combined with genetics (in vivo
tools, for example, the two hybrid system). Structural studies of transcription factors are
likely to be a gold mine for basic science and small molecule discovery for therapeutics.
Transcription Factors in Parasites
Transcription in parasites is poorly understood. The establishment of successful
transfection procedures for Leishmania [Dyann Wirth, Harvard], Plasmodium [Dyann
Wirth, Harvard; Louis Miller, NIH], Toxoplasma [John Boothroyd, Stanford] and
Trypanosomes [George Cross, Rockefeller] are likely to aid investigators in analysis of
gene expression. Scientists with expertise in transcription may discover fundamental
principles of parasite biology and shed light on host-parasite interactions. Identifying
regulatory molecules may provide clues to vaccines or develop therapeutic strategies.
Little is known about the subunit structure of RNA polymerases either from
Plasmodium or Trypanosome. Characterization of RNA polymerases and associated basal
transcription complex from these and/or other parasites are essential. Most eukaryotic
RNA polymerase II is present in two phosphorylated forms [Young RA (1991) Ann Rev
Biochem 60 689-715]. Phosphorylation is mainly located on the carboxy terminal domain
[CTD] of the largest subunit [RPB1]. The CTD of yRPB1 consists of 26 or 27 nearly
identical copies of a hepta-peptide sequence [Tyr-Ser-Pro-Thr-Ser-Pro-Ser]. In mammals
there are about 52 repeats. CTD in P. falciparum is distinct with a 63 aa tail [Li et al NAR
(1989) 17 9621-9636]. Trypanosomes lack this type of CTD [Smith et al (1989) Cell 56 815827]. We do not know whether CTD-associated factors identified in other organisms
[Koleske et al (1992) Cell 69 883-894 and Thompson et al (1993) Cell 73 1361-1375] may
interact with similar regions of the Plasmodium CTD. Unique regions of Plasmodium CTD
may be used as an affinity tag to identify molecules which interact with the region. In a
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
parallel genetic approach, yCTD may be modified to resemble Plasmodium CTD [or parts
of it] and genetic selection in yeast [Nature (1993) 364 121-126] may identify factors,
which have homologoues in Plasmodium.
For regulated transcription by pol II, the binding of TBP to the TATA sequence is
essential but structurally TBP is not universal. P. falciparum TBP [pfTBP] is bit different
[Hyde et al (1992) Nature 360 541; McAndrew et al (1993) Gene 124 165-171]. The authors
point out that such differences "could be exploited chemotherapeutically" in future. It is
of interest to note the highly conserved Proline [β sheet 1] in TBP [except Plasmodium]
which is essential for specificity of transcription by pol II as well as pol III [Schultz et al
(1992) Cell 69 697-702]. Substituting Proline abolishes transcription by pol II and pol III
but not by pol I. In pfTBP, Leucine replaces this Proline at position 50. It is exciting to
speculate the pol I bias (?) of pfTBP with reported transcription of VSG and PARP by
RNA polymerase I [Chung et al (1993) MCB 13 3734-43]. Comparison of pfTBP with
Arabidopsis thaliana TBP-2 [Nikolov et al (1992) Nature 360 40-46] reveal differences in the
region identified as the yTFIIA interaction site by mutational analysis. Upon closer
analysis of H2 and S1' the differences appear to be located mostly in H2. Given the
subunit [heterodimeric in yeast and heterotrimeric in humans] structure of TFIIA,
perhaps one subunit is more conserved across species while other(s) may be species
specific. Two-hybrid systems may be useful to search for parasite homologues. Overall,
the TBP core that interacts with DNA is better conserved than the faces, that are likely to
interact with other proteins in a complex. Solving the structure of pfTBP complexed with
its specific {?} sequence will be valuable. The canonical TATA sequence may not be the
binding site for pfTBP.
The differences in Plasmodium CTD and TBP may offer exciting clues. If pfTBPDNA interaction is kinetically equivalent to the mammalian system, we could use the
protein-DNA complex to assemble other factors in an in vitro transcription system for
studying parasite gene expression. The feasibility of this approach is substantiated from
my work (Datta, S., unpublished) using fractionated components from the HeLa system
[Buratowski S et al (1989) Cell 56 549-561]. Plasmodium CTD used as an affinity tag may
identify interacting target molecules (unique to the infected state) of therapeutic interest.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
In Trypanosomes, hybridization with TBP probes had limited success [George
Cross, personal communication] in view of differences between ‘classical’ and parasite
TBP. In Trypanosomes, two abundant messages [VSG and PARP] are transcribed by αamanitin resistant RNA polymerase in parasites containing disruption in 2 of the 4
alleles coding for the largest subunit of RNA polymerase II [Chung et al (1993) MCB 13
3734-3743]. There is little doubt that TBP interacts with most RNA polymerases [Sharp
PA (1992) Cell 68 819-821] and Trypanosome TBP identification may take the pol I route.
That TBP or TAFs may {1} play a role in DNA replication [Heintz NH et al (1992)
TIG 8 376-381], {2} participate [perhaps in cooperation with other proteins; Weinert &
Hartwell (1993) Genetics 134 63-80] in cell cycle regulation [Nature (1993) 362 175-181],
{3} form complex with TFIIH [BTF2] which is encoded by ERCC-3, a DNA repair gene
found mutated in patients with xeroderma pigmentosum [Science (1993) 260 55-63],
suggests a role of TBP in cellular processes, including repair [Science (1993) 260 53-58]
and recombination [VSG expression site and telomeric DNA repeat binding protein;
MCB (1993) 13 1306-1316]. The latter generates antigenic variation in Trypanosome VSG.
Transcriptional re-programming by TBP may regulate such variation. Identifying key
regulatory factor[s] may provide a system where transcriptional re-programming [and
hence, antigenic variation] may be controlled by an external "molecular switch" without
disturbing the general physiology of the parasite [Schena et al (1991) PNAS 88 10421-25].
Once characterized, RNA polymerase subunit[s] and transcription factors may be
mutated and transfected in parasites containing disruption of the chromosomal gene
[Steven Beverly, Leishmania; Lex van der Ploeg, Trypanosome]. Developing tools to use
suppressor genetics in parasites may further aid in dissecting the transcription complex.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
PRE-PROPOSAL
Introduction
Parasitic diseases are a major cause of mortality and morbidity worldwide. In the
tropics, it causes about 200 million new infections and claims more than 2 million lives,
annually. In 1992, there were about 800,000 child deaths due to malaria in Africa [1]. The
re-emergence of opportunistic parasitic infections in the affluent industrialized nations
are increasing with the spread of AIDS.
Diseases caused by animal parasites afflicted our ancestors. Ancient documents
from China, Assyria and India describe high intermittent fevers, typical of malaria.
During 5th century B.C., Hippocrates characterized the fevers and symptoms of malaria
while Aristotle wrote about [the cysticercus stage of] a certain worm [Taenia solium] in
the muscles of pigs. Probably, the first scientific report of a parasitic human infection
[caused by the intestinal tapeworm Diphyllobothrium latum] was documented in 1602 by
Plates, a Swiss physician [2].
Malaria still presents the single greatest threat in the tropics. In 1880, Laveran
described the asexual stages of Plasmodium falciparum, the causative agent of human
malaria. In 1898, Sir Ronald Ross, working in Calcutta (India), showed that the parasite
developed in the mosquito and was transmitted by the bite of the insect [3]. Almost
immediately, malaria control became synonymous with mosquito control. Ross and
Laveran received the Nobel Prizes for their seminal contribution.
Treatment of malaria has not changed much since the 17th century, when the
Peruvian bark, cinchona, was first used as chemotherapy. Quinine and cinchonine, the
alkaloids of the cinchona tree, were isolated in 1820 and synthetic anti-malarials were
developed in Germany [Pamaquine in 1924 and Chloroquine in 1934], England
[Proquanil in 1944] and USA [Primaquine in 1952]. Prevention of malaria by mosquito
control gained impetus with the development of the insecticide DDT. However, DDT
has the potential to wreck the ecological balance and promote other disasters.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
In 1955, to control a malaria epidemic in Borneo, the World Health Organization
[WHO] sprayed the country with DDT which killed mosquitoes and houseflies. The
dead houseflies were consumed by geckos, which in turn died. Without the geckos to
prey upon them, Borneo's rat population exploded and resulted in a bubonic plague
epidemic. To contain the plague epidemic, WHO air-dropped thousands of cats on the
island ["the day it rained cats"]. Several thousand additional mortalities resulted from
the second [plague] epidemic before the cats finally brought the rat population under
control.
Molecular biology and biotechnology may help produce a malaria vaccine but it
is still a work in progress. One claim of a "highly effective" immuno-therapy was only
effective in one-third of patients tested [4]. However, parasitology is not completely
devoid of molecular milestones. In 1976, Trager and Jensen succeeded to grow human
malaria parasites in continuous culture [5]. Transfection procedures were established for
Trypanosomes [6], Leishmania [7], Plasmodium [8] and Toxoplasma [9]. Successful gene
replacement and disruption has been achieved in Leishmania [10] and Trypanosome [11].
Using an oligonucleotide screening strategy, the equivalent of the human and yeast
TATA binding protein [TBP] was isolated from Plasmodium falciparum [12]. Recently the
isolation of a red blood cell [erythrocyte] receptor used by Plasmodium to invade the cell
was reported [13].
The isolation of TBP from malaria parasite and establishment of transfection
procedures for Plasmodium, represent steps toward understanding transcription in
Plasmodium since TBP is essential to transcription in eukaryotes [14]. Proliferation of the
parasite in the host is dependent on its ability to initiate transcription of the essential
genes and direct its asexual stages of development.
After a mosquito bites, the salivary fluid containing sporozoites [motile forms of
the parasite] are delivered in the blood. Infection initiates following entry of sporozoites
in liver parenchymal cells where parasites divide asexually. For P. falciparum, within 5-7
days, each infected liver cell releases about 40,000 progeny [merozoites] which invade
red blood cells and initiate the erythrocytic phase of infection. Within the erythrocyte,
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
the parasite grows to form early trophozoite, followed by asexual division to become a
schizont composed of merozoites. The erythrocytic cycle completes with rupture of the
erythrocyte within 48 hours and release of merozoites, which proceed to invade other
erythrocytes. The cycle continues with tertian periodicity [48 hours] unless intervened.
Antibodies directed against stage-specific antigens were mostly ineffective due to
the intra-cellular location of parasite. Chemotherapy aimed at retarding proliferation of
parasites adversely affects normal cells. The mechanism by which the parasite attaches
itself to the erythrocyte, and subsequently gains entry, may be better understood with
the recent identification of the receptor [13]. However, the ability of the parasite to
multiply in the liver and erythrocyte presents a key threat.
Universally, almost all biological processes are mediated through the action of
proteins. Transcription of DNA is the first step leading to protein synthesis. Initiation of
transcription is a complex process. Biochemical fractionation of extracts made from
mammalian cells and mutational analysis combined with suppressor genetics in yeast,
have yielded a wealth of information. TBP, isolated as a key component of basal
transcription by RNA polymerase II, is a common denominator in transcription by all
three eukaryotic RNA polymerases [14]. Most subunits of yeast RNAPII have been
cloned and sequenced [15]. TBP, along with other basal transcription factors [mostly
specific for the type of RNAP] and RNAP are necessary components of the minimal
transcription apparatus [16].
Comparison of the largest subunit [RPB1] of Plasmodium RNAPII with yeast and
human RPB1, revealed striking homology and diversity in the conserved carboxy
terminal domain [CTD] of RPB1 [15]. Biochemical analysis and suppressor genetic
studies in yeast have identified transcription factors which interact with the CTD [17,
18]. TBP from Plasmodium, plant, yeast, frogs and humans show homology in several
region but Plasmodium TBP diverges in a region that map to a RNAP interaction site and
in another region that may interact with TFIIA, a RNAPII basal transcription factor [14].
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
The structural genes of Plasmodium may be transcribed by RNAPII. However, a
similar notion held for VSG genes was dispelled when VSG transcription was unaffected
in Trypanosomes with chromosomal disruptions in two RNAPII alleles [11]. Irrespective
of which RNAP is involved, Plasmodium TBP may form complexes with other RNAP
basal factors, providing a common biochemical theme. The homology and diversity of
Plasmodium CTD offers a biochemical probe for isolation of Plasmodium-specific CTD
interacting factors that may regulate transcription and gene expression.
Objective
The primary objective is to identify Malaria parasite [Plasmodium falciparum]
specific transcription factors, which may serve as targets for therapeutic intervention or
provide clues for designing novel vaccine strategies for Malaria. However, within the
two year period specified in the grant, it may be feasible only to undertake the most
direct and defined aspects of this proposal, as follows:
1.
Identifying and characterizing protein[s] that interact with the Plasmodium CTD.
2.
Identifying and characterizing protein[s] that interact with the Plasmodium TBP.
3.
Cloning the genes of proteins that interact with the Plasmodium CTD and TBP.
Hypothesis
Transcription factors regulate and maintain host-parasite interactions. Survival
of the parasite necessitates such proteins to provide common functions to "bridge" the
host-parasite functional chasm. Characterization of proteins involved in such "crosstalk" may reveal novel targets for small molecule drug discovery geared toward
therapeutic intervention. Selective disruption of the parasite-specific component may
abrogate the host-parasite interaction without adversely affecting the host physiology.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
Rationale
[1]
The sequence of RNAPII subunits are conserved in evolution. The three largest
subunits [RPB1, RPB2, RPB3] are homologues of bacterial RNAP and their sequences are
conserved from bacteria to humans [15]. In most eukaryotes, the C-terminal domain
(CTD) of RPB1 contain a consensus hepta-peptide repeat sequence [Tyr-Ser-Pro-Thr-SerPro-Ser]. The sequence is conserved amongst species and the number of repeats increase
with the complexity of the organism [yeast = 27, mouse = 52]. Yeast cells with truncated
CTD containing fewer than 10 repeats are non-viable. The CTD of yeast RPB1, interacts
with RNAPII transcription factors [17, 18]. The sequence of Plasmodium CTD shares
homology with other organisms for about 15 repeats and then diverges to form a 63 aa
tail with no homology to any published sequence [15]. This feature may be the
hypothetical "bridge" and/or a template for "cross-talk" in host-parasite interactions.
In other words, the conserved region of the Plasmodium CTD "acts" like the host
[human] CTD and hence utilizes the host functions for transcription. The unique region
of Plasmodium CTD may be specific for the biology of the parasite. This, therefore, is a
target with no cellular counterpart. Extrapolating from functional conservation, this
unique region probably interacts with one or more [transcription] factors, perhaps, in a
manner similar to the interaction of transcription factors with yCTD [17, 18]. Identifying
factors that interact with Plasmodium CTD [factors specific for the unique region] may
offer Plasmodium-specific targets for therapeutic intervention.
[2]
Kinetic studies reveal that the first, necessary and essential, step in the formation
of a pre-initiation complex for transcription initiation by RNAPII, is the sequencespecific binding of TBP [TATA binding protein] to a TATA-containing promoter [19].
But, RNAPII mediated transcription from TATA-less promoters also contain TBP in the
pre-initiation complex [14]. In addition, transcription initiation complexes formed at
promoters transcribed by RNAPI and RNAPIII contain TBP, attesting to the universal
requirement of TBP in eukaryotic transcription. The isolation of Plasmodium TBP makes
it possible to study the biochemical assembly of the pre-initiation complex with TBP
anchored to TATA sequence, as the first committed step. However, it may be argued,
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
given the A+T rich genome of Plasmodium, that TATA sequences may be arbitrary. The
latter raises doubts about the specificity of the TBP-TATA interaction in Plasmodium.
However, the following two observations may be cited in favour of the TBP interaction:
[A] During the stepwise assembly of the pre-initiation complex on a TATAcontaining promoter, the TATA sequence is bound by TBP and several other TAFs or
TBP associated factors [14]. The TBP-TAF complex is referred to as TFIID. The TATATFIID complex binds TFIIA [RNAPII basal transcription factor] to form the DA complex.
In the third step, TFIIB binds to the DA complex to form the DAB complex. A preformed
TFIIF-RNAPII complex associates with the DAB complex, followed by association with
TFIIE and TFIIH [20]. Therefore, the kinetics of binding of TBP to the TATA sequence
may be subject to modification by one or several associated proteins. While some of the
protein-protein contacts with TBP have been mapped, the list is still growing. However,
the most striking divergence of Plasmodium TBP from other TBPs, coincides with TFIIA
contact points which maps in helix 2 [H2] of TBP [21]. Hence, is there a Plasmodium
specific TFIIA that helps modify TBP-TATA specificity? By conserving some features of
the host pre-initiation complex but modifying others, the parasite may have evolved a
transcriptional machinery that is compatible with yet distinct from the host. Using
Plasmodium TBP as a “hook” it may be possible to identify associated parasite-specific
factors. The latter may be direct targets or provide clues for novel therapeutics.
[B] TATA-TBP DNA-protein interaction is only one of many possible interactions
of TBP [14]. In RNAPI and RNAPIII interactions, TBP interacts with other proteins or
protein-DNA complex. Plasmodium TBP may interact with accessory proteins and the
resulting protein-protein complex, then, may bind with sequence specificity to DNA. It
is of interest to note that a highly conserved Proline residue is located in β sheet 1 of all
TBPs except Plasmodium TBP [12, 21]. By mutational analysis in yeast, it has been shown
that replacing this Proline with any other amino acid abolishes the ability of the mutant
TBP to sustain transcription by RNAPII and RNAPIII in vivo [21] but the mutant TBP
remains functional for RNAPI transcription in vivo. It will not be without precedent if
some Plasmodium mRNAs may be RNAPI transcripts, similar to transcription of VSG
genes in Trypanosome [11].
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
Methods
A.
Preparation of Extracts
B.
CTD and TBP Affinity Chromatography
C.
DNA Binding Specificity of Plasmodium TBP
[A] Preparation of Extracts
Large-scale cultivation of the erythrocytic stage of P. falciparum is an established
procedure and several methods exist in the literature to make protein extracts [22]. The
procedures used for making whole cell and nuclear extracts from mammalian cells will
be tested [23]. Extract suitability will be tested by reproducible retention of specific
proteins by affinity chromatography and analytical gel electrophoresis to estimate
integrity of proteins. To help enrich for any protein that may be retained on the affinity
columns, each batch of extract will be fractionated over DEAE and CM matrices and
eluted with step gradients. Individual fractions will be concentrated and applied to CTD
and TBP affinity columns.
[B] CTD and TBP Affinity Chromatography
Several protocols exist to identify interacting proteins [24]. In addition, GST
fusions or similar methods exist where the base protein [GST] is attached to the bead
while the fusion [CTD] is free to interact [25]. Plasmodium CTD will be subcloned
independently or as a fusion and CTD or CTD-fusion purified to homogeniety. This will
be immobilized on beads to serve as the CTD affinity column. To select proteins that
bind to the unique region of the CTD, the repeats will be deleted in the expression clone
or the unique peptide will be synthesized. In the latter case, reversible photoactivated
cross-linkers may be used to identify proteins that interact with the peptide [26]. Yeast
CTD will serve as control. Similar strategy will be followed for Plasmodium TBP [gift of
John Hyde, UMIST]. Retained proteins will be sequenced and used for cDNA screening.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
[C] DNA Binding Specificity of Plasmodium TBP
TATA oligonucleotides and several mutant combinations [gift of Kevin Struhl,
Harvard] will be used in gel retardation assays to determine the sequence and binding
specificity of Plasmodium TBP, if any [27]. If specific binding is detected, then partially
fractionated Plasmodium extracts will be tested to determine if higher order complexes
[pre-initiation complexes?] can form on such a template [20].
Conclusion
Knowledge about RNAPII and TBP obtained from other eukaryotes [humans,
yeast] is the key to frame important medically relevant questions about the malaria
parasite, Plasmodium falciparum, and its interaction with humans. Characterization of
transcription factors that mediate parasite-specific functions are of medical significance.
The simplicity of this approach may unambiguously identify proteins of interest. The
quest for therapy is based on understanding, identifying and disrupting these key
molecule[s] involved in the biology of the host-parasite interaction.
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
References
1.
WHO. 1992. World malaria situation. WHO Stats Quarterly 45 257-66
2.
Katz, M., Despommier, D.D. and Gwadz, R. 1982. Parasitic Diseases. SpringerVerlag, New York.
3.
Ross, R. 1898. Report on the cultvation of Proteosoma Labbe in grey mosquitoes.
Government Press, Calcutta.
4.
Valero, M.V. et al. 1993. Vaccination with SPf66, a chemically synthesized
vaccine, against Plasmodium falciparum malaria in Colombia. Lancet 341 705-10
5.
Trager, W. and Jensen, J.B. 1976. Human malaria parasites in continuous culture.
Science 193 673-5
6.
Bellofatto, V. and Cross, G.A.M. 1989. Expression of a bacterial gene in a
trypanosomatid protozoan. Science 244 1167-9
7.
Laban, A. and Wirth, D.F. 1989. Transfection of Leishmania enrietti and
expression of choramphenicol acetyltransferase gene. Proc. Natl. Acad. Sci. USA
86 9119-23
8.
Goonewardene, R., Daily, J., Kaslow, D., Sullivan, T.J., Duffy, P., Carter, R.,
Mendis, K. and Wirth, D.F. 1993. Transfection of the malaria parasite and
expression of firefly luciferase. Proc. Natl. Acad. Sci. USA 90 5234-6
9.
Soldati, D. and Boothroyd, J.C. 1993. Transient transfection and expression in the
obligate intracellular parasite Toxoplasma gondii. Science 260 349-52
10.
Cruz, A. and Beverly, S.M. 1990. Gene replacement in parasitic protozoa. Nature
348 171-4
11.
Chung, H.M., Lee, M.G., Dietrich, P., Huang, J. and Van der Ploeg, L.H. 1993.
Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.
Mol. Cell Biol. 13 3734-43
12.
McAndrew, M.B., Read, M., Sims, P.F.G. and Hyde, J.E. 1993. Characterisation of
the gene encoding an unusually divergent TATA-binding protein (TBP) from the
extremely A+T-rich human malaria parasite Plasmodium falciparum. Gene 24
165-171
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
13.
Horuk, R., Chitnis, C.E., Darbonne, W.C., Colby, T.J., Rybicki, A., Hadley,
T.J. and Miller, L.H. 1993. A receptor for the malarial parasite Plasmodium vivax:
The erythrocyte chemokine recptor. Science 261 1182-4
14.
Hernandez, N. 1993. TBP, a universal eukaryotic transcription factor? Genes and
Dev. 7 1291-1308
15.
Young, R.A. 1991. RNA Polymerase II. Ann. Rev. Biochem. 60 689-715
16.
Parvin, J.D. and Sharp, P.A. 1993. DNA topology and a minimal set of basal
factors for transcription by RNA polymerase II. Cell 73 533-40
17.
Koleske, A.J., Buratowski, S., Nonet, M. and Young, R.A.1992. A novel
transcription factor reveals a functional link between the RNA polymerase II
CTD and TFIID. Cell 69 883-94
18.
Thompson, C.M., Koleske, A.J., Chao, D.M. and Young, R.A. 1993. A
multisubunit complex associated with the RNA polymerase II CTD and TATAbinding protein in yeast. Cell 73 1361-76
19.
Fire, A., Samuels, M. and Sharp, P.A. 1984. Interactions between RNA
polymerase II, factors and template leading to accurate transcription. J Biol.
Chem. 259 2509-16
20.
Buratowski, S., Hahn, S. Guarente, L. and Sharp, P.A. 1989. Five intermediate
complexes in transcription initiation by RNA polymerase II. Cell 56 549-61
21.
Nikolov, D.B., et al. 1992. Crystal structure of TFIID TATA-box binding protein.
Nature 360 40-46
22.
Hyde, J.E. ed. 1993. Protocols in Molecular Parasitology. Humana Press, NJ.
23.
Wu, R., Grossman, L. and Moldave, K. ed. 1983. Methods in Enzymology 101
568-598
24.
Guarente, L. 1993. Strategies for the identification of interacting proteins. Proc.
Natl. Acad. Sci. USA 90 1639-41
25.
Truant, R., Xiao, H., Ingles, C.J. and Greenblatt, J. 1993. Direct Interaction
Between the Transcriptional Activation Domain of Human p53 and the TATA
Box-binding Protein. J. Biol. Chem. 268 2284-7
26.
Radeke, M.J. and Feinstein, S.C. 1991. Analytical purification of the slow, high
affinity NGF receptor: identification of a novel 135 kd polypeptide. Neuron 7
141-150
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
27.
Singer, V.L., Wobbe, C.R. and Struhl, K. 1990. A wide variety of DNA
sequences can functionally replace a yeast TATA element for transcriptional
activation. Genes and Dev. 4 636-45
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
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Molecular Medicine: Statement of Research & Pre-proposal for Transcription Targets in Molecular
Parasitology
Summary of Education & Positions
1976
School Leaving Certificate [GCE]
University of Cambridge, Cambridge, UK [Local Examinations Syndicate]
1980
BSc [Honours] Biochemistry and Medical Physiology
Minors: Physics and Chemistry
Presidency College, University of Calcutta, Calcutta, India
1988
PhD [Molecular Biology and Genetics]
Rutgers University School of Medicine & collaboration with Princeton University
1989-1991
Research Fellow in Medicine
Harvard University & Harvard Medical School; Massachusetts General Hospital
1991-present
Research Associate
Whitehead Institute, Massachusetts Institute of Technology
1991-present
Instructor in Medicine
Harvard Medical School
Proposed - 1994
Research Scientist
University of California, Berkeley and
University of California, San Francisco
UCSF School of Medicine
CONTACT INFORMATION
Dr Shoumen Datta
Whitehead Institute for Biomedical Research
Massachusetts Institute of Technology
Cambridge, Massachusetts 02142-1479
Tel:
Fax:
Eml:
[617] 252-1940
[617] 252-1902
shoumen@mit.edu
27 September 1993 Dr Shoumen Datta, Whitehead Institute, Massachusetts Institute of Technology (MIT)
15
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
ABSTRACT
1
Regulation of transcription initiation provides a key mechanism for control of gene
expression. Basal transcription initiation factors, best characterized in mammals, and RNA
polymerase II, which is best understood in yeast, are two important components of the transcription
apparatus. The objective of this research is to identify and characterize the components of the basal
transcription apparatus from yeast. This would allow us to employ the powerful combination of yeast
genetics and biochemistry to advance our understanding of basal transcription initiation. As a first
step, we aim to fractionate yeast nuclear extracts in order to reconstitute an in vitro transcription
system and to analyze the assembly of initiation complexes by native gel electrophoresis.
Information and reagents available from fractionation of mammalian extracts are likely to aid in
identifying the yeast homologues of the basal transcriptional machinery. In the next step, we plan to
purify and clone basal transcription initiation factors that are unavailable. The recombinant factors
will be used to replace one or more fractions in in vitro transcription. The information obtained from
these experiments will improve our understanding of the basal transcription initiation apparatus and
may provide clues to the mechanisms by which the basal transcription apparatus is regulated.
SPECIFIC AIMS
To improve our understanding of the mechanisms of eukaryotic transcription initiation, the project
aims to:
1)
fractionate yeast nuclear extracts to obtain highly purified components of the basal
transcription initiation apparatus to reconstitute an in vitro transcription system capable of
accurate initiation;
2)
identify basal transcription initiation factors in yeast that have mammalian counterparts by
using a gel retardation assay;
3)
purify transcription factors to homogeneity and clone and express their genes.
RELATIONSHIP TO THE CANCER PROBLEM
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
Cancer results from aberrant gene expression. Regulation of transcription initiation provides
2
a key mechanism for control of gene expression. Yet the basic process of transcription initiation and
the mechanisms that regulate this process are poorly understood. In this project we propose to
characterize the components of the basal transcription initiation apparatus in eukaryotes. A well
defined transcription initiation process will provide the foundation to investigate the mechanism by
which deregulation of this process can cause cancer. Protein products of several oncogenes (FOS,
JUN, MYC, etc.) are important regulatory transcription factors which probably act at the level of
transcription initiation to deregulate normal cellular processes. Information obtained by comparing
normal proteins with their oncogenic counterparts, will aid in improved drug design and effective
therapy.
INTRODUCTION
The process of transcription initiation by RNA polymerase II is complex, yet we have some
clues [4, 29, 38, 41, 50]. Components of the transcription initiation apparatus are currently (1991) the
focus of considerable attention. However, basal transcription initiation factors in yeast are largely
unidentified, despite a relatively detailed picture of yeat RNA polymerase II. The availability of yeast
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
3transcription factors would allow us to combine the power of yeast genetics and biochemistry to
understand the details of the process of transcription initiation.
In the following sections we present an overview of the field and the rationale to pursue the
proposed research:
A.
The basal transcription apparatus and initiation of transcription
B.
The components of the basal transcription apparatus
1. TFIID
2. TFIIA
3. TFIIB
4. TFIIF, TFIIE
5. TFIIG, TFIIH
6. RNA polymerase II
C.
Developing a reconstituted yeast transcription system
D.
Suitability of the laboratory environment for this project
The basal transcription apparatus and initiation of transcription
Extracts made from mammalian cells [35] or nuclei [12] support in vitro transcription of
TATA-containing templates. To understand the nature of the basal transcriptional machinery, it was
necessary to isolate the individual components. Chromatographic fractionation of HeLa cell nuclear
extracts and reconstitution of basal transcription in vitro indicated that multiple components are
contained within the basal transcription apparatus [37, 40, 47]. Kinetic studies showed that the first
committed event in transcription initiation is the sequence-specific binding of TFIID (Transcription
Factor, RNA polymerase II, fraction D) to a TATA-containing promoter sequence [11, 15, 46].
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
Our understanding of the stepwise assembly of basic transcription apparatus in mammalian
4
cells is based on Buratowski et al. [1]. Using partially purified transcription factors and a gelretardation assay, the authors demonstrated the sequential assembly of the transcription apparatus on a
minimal promoter [1]. In summary, the key event is the binding of TFIID to the TATA element.
TFIIA, which is unable to bind to DNA by itself, binds to the TFIID-DNA complex to generate the
DA complex. Addition of TFIIB creates the DAB complex which associates with a preformed TFIIFRNA polymerase II macromolecule followed by association with TFIIE [9, 18]. TFIIG [44] and
TFIIH [30] have been identified in pre-initiation complex. TFIIG associates with the complex prior to
the DABF-polII step [44] and TFIIH is likely to enter the complex either subsequent to or with TFIIE
[30].
The components of the basal transcription apparatus
TFIID
From fraction TFIID, a single protein was identified which binds to the TATA sequence.
Yeast TFIID is a 27 kD protein. The TFIID gene is essential for yeast viability [14] and it has been
cloned from humans [32], yeast [24, 27, 28, 42], drosophila [26] and plant [19]. The protein is
strikingly conserved in its C-terminal 180 aa (between various species) while containing a highly
variable N-terminal domain. Other proteins have been reported to associate with TFIID and may
influence transcription [13].
TFIIA
The second step in the formation of the transcription complex requires TFIIA [1]. A 35-38 kD
protein is the essential component of wheat and human TFIIA [5]. TFIIA from yeast or wheat can
replace human TFIIA in a functional assay [5, 23]. Yeast TFIIA is a heterodimeric protein, both
subunits of which have been cloned (S. Hahn, personal communication). TFIIA can be purified by
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
5TFIID affinity chromatography [45] and it may associate with TFIID independent of DNA in vivo
[1, 23].
TFIIB
In step three, DAB complex formation requires fraction TFIIB. A 30 kD protein is the
essential component of TFIIB and the human TFIIB gene has been cloned [22].
TFIIF, TFIIE
The DAB apparatus associates with a preformed TFIIF-RNA polymerase II complex [9, 18].
Subsequently TFIIE enters the complex [30]. Slower migrating complexes containing these factors
can be resolved by native gel electrophoresis [1]. Human TFIIF consists of 30 kD and 74 kD
subunits, originally identified as RNA polymerase associated proteins (RAP30, RAP74) [6, 17, 43].
Human TFIIE is a hetero-dimeric protein with 34 kD and 56 kD subunits [30, 39].
TFIIG, TFIIH
Mammalian transcription complexes have been reported to contain TFIIG [44] and TFIIH
[30]. TFIIG is required for preinitiation complex formation prior to association of RNA polymerase
II. It shares functional similarities with, but is chromatographically distinct from, TFIIA [44]. TFIIH
enters the complex either with or subsequent to TFIIE [30].
RNA Polymerase II
An essential component of the basal transcription apparatus is RNA polymerase II [41, 50].
The enzyme alone is incapable of accurate transcription initiation. Yeast RNA polymerase II is a
complex enzyme containing 11 subunits [50 and references therein]. The yeast subunit genes have all
been cloned. The three largest subunits are homologues of the prokaryotic RNA polymerase core
subunits. Some of the human RNA polymerase II subunit genes have been cloned and sequence
analysis reveals that they are closely related to their yeast counterparts. A unique feature of the largest
subunit of eukaryotic RNA polymerase II (RPB1) is the presence of a repeated hepta-peptide at its
carboxy-terminal domain (CTD). The hepta-peptide repeat sequence is highly conserved amongst
species and the number of repeats increases with the complexity of the organism. The CTD appears to
have a role in transcription initiation [10, 50].
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American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
6
Developing a reconstituted yeast transcription system
Fractionation of mammalian nuclear extracts and isolation of some of the human transcription
factor genes have improved our understanding of transcription initiation [4, 41]. Detailed
characterization of transcription factors is limited by the lack of an efficient genetic approach in
mammals. For this purpose we have chosen to develop a yeast system which would provide the power
to combine biochemical studies and genetic characterization. The high degree of conservation of the
transcriptional process [5, 7, 8, 20, 21, 23, 31, 36] suggests that models derived from studies on yeast
will be applicable to higher eukaryotes.
Transcription competent yeast nuclear extracts have been reported [33] and a preliminary
fractionation scheme has been published [16]. This fractionation scheme does not allow investigators
to relate the yeast fractions to transcription initiation factors already described in the mammalian
system. Establishing a comparable system in yeast is still necessary.
We plan to use reconstitution of in vitro transcription activity and the gel retardation assay [1]
to monitor separation of individual components and assign yeast functional homologues of the
mammalian basal transcription initiation factors. A few components of the basal transcription
apparatus are at hand from the yeast system: the cloned yeast TFIID gene and purified recombinant
yeast TFIID and various mutant versions of the protein (S. Buratowski), cloned yeast TFIIA genes (S.
Hahn, personal communication); cloned yeast RNA polymerase II subunit genes, purified yeast RNA
polymerase II holoenzyme (S.-M. Liao, S. Buratowski and R. Young). In addition, work in our
laboratory is underway to clone yeast TFIIB and TFIIF genes using mammalian gene probes (S.-M.
Liao, J. Zhang, S. Buratowski and R. Young).
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American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
7
In contrast to the mammalian in vitro transcription system, which was developed more than a
decade ago [35], the yeast in vitro system is relatively new [33, 48] and still undergoing modifications
[49]. Information obtained from extensive fractionation of HeLa cell nuclear extracts by a number of
laboratories over several years made it possible for Buratowski et al [1] to demonstrate the assembly
of intermediate complexes of a transcription initiation apparatus. Fractionation of yeast nuclear
extracts is still in its preliminary stages [16]. Without a reproducible fractionation scheme and assays
to monitor in vitro transcription initiation and assembly of initiation complexes, it will be difficult to
dissect the individual components which make up the basal transcription apparatus.
The objective of this research is to identify individual components of the yeast basal
transcription apparatus and to develop reconstituted in vitro transcription system. It is expected that
this system will be used in combination with a genetic analysis of RNA polymerase II and
transcription factor genes. Mutant proteins can then be tested functionally both in vivo and in vitro.
EXPERIMENTAL APPROACH
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
The power of resolution of column chromatography makes this method key to the
8
identification and purification of basal transcription initiation factors. Yeast nuclear extracts will be
fractionated and assayed for transcriptional activity in vitro by reconstituting various fractions. Active
fractions will be used for assembly of initiation complexes and analyzed by the gel retardation assay
[1]. Genetic methods and gene cloning subsequent to protein purification will be employed. The
experimental approach is discussed in subsections as follows:
A.
Yeast nuclear extracts
B.
Fractionation and reconstitution of in vitro transcription
C.
Gel retardation assay for assembly of initiation complex
D.
Preparative purification of transcription factors
E.
Transcription factors identified by genetic methods
Yeast nuclear extracts
Preparation of highly active nuclear extracts capable of supporting transcription in vitro is
essential to any fractionation scheme. We have made several important modifications to the original
yeast nuclear extract protocol [33]. To allow cells to recover adequately from cell wall digestion, the
regeneration time has been increased. Nuclei were extracted with ammonium sulfate and
contaminating nucleic acids were removed by DEAE-Sephacel treatment [34]. The supernatant was
precipitated with solid ammonium sulfate and the recovered protein pellet was resuspended and
dialyzed. These modifications resulted in substantial improvement in the quality of the in vitro
transcription signal.
Fractionation and reconstitution of in vitro transcription
Yeast nuclear extracts have been chromatographed over DEAE Sepharose and step-eluted
with increasing salt concentration. The fractions will be assayed in various combinations to
reconstitute transcription activity in vitro. The active fractions from this step will be subjected to
further rounds of ion exchange and molecular sieve chromatography to enrich for transcription
factors. Reconstitution assays will be performed with each fraction and subfraction to continually
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
9optimize for transcriptional activity. We have purified yeast RNA polymerase II and purified
recombinant yeast TFIID to supplement in vitro reconstitution assays, as appropriate. These
experiments will allow us to identify the yeast homologues of the mammalian basal transcription
initiation factors. With increased fractionation, the yeast system may identify additional basal
transcription initiation factors. The ultimate goal is to use these purified factors to establish a
standardized reconstituted in vitro transcription system capable of accurate initiation.
Gel retardation assay for assembly of initiation complex
The reconstitution assay described above identifies active fractions that are sufficient and
necessary for basal transcription but lacks in its ability to assign yeast homologues of the mammalian
system. To identify which yeast fractions contain the functional equivalent of the previously
identified mammalian basal transcription factors, the gel retardation assay will be used [1]. In brief,
purified recombinant yeast TFIID and the same promoter used for in vitro reconstitution assays will
be used to generate the first complex. To this we will add transcriptionally active fractions and
monitor the assembly of initiation complexes. It is likely that the yeast pattern of the transcription
intermediate complexes will resemble previously identified mammalian complexes and allow us to
tentatively identify the yeast homologues of the mammalian factors. To confirm the identity of these
homologues we will pursue further experiments.
Preparative purification of transcription factors
Having established a functional fractionation scheme we will isolate preparative amounts of
initiation factors. Information from protein micro-sequencing will help in the design of
oligonucleotide probes for PCR and hybridization to cDNA libraries to clone the transcription factor
genes.
Yeast cells may contain limited copies of some basal transcription factors, and we may find
that it is difficult to obtain sufficient amounts of protein from fractionation of nuclear extracts. As an
alternative approach, we could use yeast whole cell extracts (yWCE) [49]. The latter extract is easier
to prepare and can be scaled up to a greater degree than preparation of nuclei. Though initial yWCE
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
10preparations were less active than nuclear preparations, recent improvements generate yWCE that are
highly active [49] and it was successfully used as a source to purify yTFIIA [23].
Transcription factors identified by genetic methods
Genetic suppressor analysis has led to the identification of genes whose products may interact
with TFIID (S. Buratowski and H. Zhou, personal communication) and/or the largest subunit of RNA
polymerase II (A. Koleske, C. Thompson and R. Young, personal communication). The protein
products of these suppressor genes may interact with the basal transcription machinery in vivo. We
have produced recombinant proteins using the cloned suppressor genes and will determine whether
these proteins are active in the in vitro reconstitution assay and the initiation complex assembly assay.
CONCLUDING REMARKS
Establish a reconstituted in vitro transcription system using purified yeast basal transcription
factors, in a manner that will allow us to relate the yeast components to the mammalian basal
transcription initiation factors. The completion of the proposed project will advance our
understanding of the process of transcription initiation. It will provide a foundation for identifying
biochemical targets within the transcription apparatus which respond to activators and repressors of
transcriptional activity.
EUKARYOTIC RNA POLYMERASE II TRANSCRIPTIONAL RECONSTITUTION IN VITRO
American Cancer Society _ Dr Shoumen Datta, Whitehead Institute, MIT <1991> shoumen@mit.edu
11
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