The activation of oncogenic cellular genes involved in transformation and of many other genes seems to be, at least in some cases, at the level of transcription. This proposal centers on the analysis of initiation of transcription by RNA polymerases II and III in vitro systems which faithfully reproduce the site and requirements for in vivo initiation of transcription. We will use a combination of straight biochemical fractionation approaches with a genetic approach which takes advantage of template and transcriptional machinery mutants as well as nucleotide analogs. The RNA polymerase II work will focus on the proteins involved in initiation by analyzing in detail the biochemical (enzyme with cofactors) as well as genetic (i.e., in the template sequence) features required for the formation of the transcription initiation complex. We will use transcriptional extracts from HeLa cells and templates consisting of oncogenic adenovirus DNA inserted in bacterial plasmids or phages. Novel features of the initiation process, like for example, unwinding of the DNA double helix or the mechanism of action of the inhibitor of transcription initiation, DRB, will be some of the partial processes that will be analyzed in detail in transcriptional initiation complexes. The unwinding assay will also be used for the VAI RNA genes transcribed by RNA polymerase III. The formation of a transcriptional complex, which distinguishes between the initiation and elongation phases of transcription, revealed a large molar excess of initiation events. This excess could be related to a mechanism of gene activation by an increase in the amounts of elongation factors or might be an artifact of the in vitro system. Point mutants in the DNA template will be generated by site-specific mutagenesis on M-13 cloning vectors which allow rapid sequence analysis of the mutants and in vitro transcription with the replicative form of the recombinant DNA phage. Site-specific point mutants allow to better establish the role of each element of the promoter sequence, since the environment around the mutation remains constant. The interactions between the mutant or wild-type promoter sequences and the elements of the transcriptional machinery, cellular RNA polymerase II Alpha-amanitin resistant mutants and transcriptionally resistant DRB resistant mutants will be analyzed. Mutations in the RNA polymerase or factors should produce some abnormal interactions. We will continue our efforts in establishing the mechanism of action of the transcription initiation inhibitor DRB and the biochemical basis for these cell mutants.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI013231-11
Application #
3125417
Study Section
Virology Study Section (VR)
Project Start
1976-06-30
Project End
1989-06-30
Budget Start
1987-12-01
Budget End
1989-06-30
Support Year
11
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Wistar Institute
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Clark, R F; Cho, K W; Weinmann, R et al. (1991) Preferential distribution of active RNA polymerase II molecules in the nuclear periphery. Gene Expr 1:61-70
Caceres, J; Glikin, G; Bravo, R et al. (1990) c-fos mediated stimulation of an AP-1 DNA binding activity in undifferentiated teratocarcinoma cell lines. Oncogene 5:59-67
Costa-Giomi, P; Caro, J; Weinmann, R (1990) Enhancement by hypoxia of human erythropoietin gene transcription in vitro. J Biol Chem 265:10185-8
Saltzman, A G; Weinmann, R (1989) Promoter specificity and modulation of RNA polymerase II transcription. FASEB J 3:1723-33
Lazard, D; Fernandez-Tomas, C; Gariglio, P et al. (1989) Modification of an adenovirus major late promoter-binding factor during poliovirus infection. J Virol 63:3858-64
Zandomeni, R; Zandomeni, M C; Weinmann, R (1988) A rapid purification method for calf thymus casein kinase II. FEBS Lett 235:247-51
Rappaport, J; Cho, K; Saltzman, A et al. (1988) Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II. Mol Cell Biol 8:3136-42
Zandomeni, R; Zandomeni, M C; Shugar, D et al. (1986) Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription. J Biol Chem 261:3414-9
Cho, K W; Khalili, K; Zandomeni, R et al. (1985) The gene encoding the large subunit of human RNA polymerase II. J Biol Chem 260:15204-10