A Purified Adenovirus 289-Amino-Acid ElA Protein Activates RNA Polymerase III Transcription In Vitro and Alters Transcription Factor TFIIIC
Author(s)HARTER, MARIAN L.; WANG, DUEN-MEI; SOONG, CHU-JING; DATTA, SHOUMEN
We have previously demonstrated that a purified bacterially synthesized ElA 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase UI. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VAT transcription by sequestering TFIIIC. However, the addition of purified ETA to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VAT transcription. When included in the same reaction, an ElA-specific monoclonal antibody reverses the restoration. Incubation of purified ETA with either HeLa cell nuclear or whole-cell extracts alters the DNA-binding properties of TFIIIC as detected by gel shift assays. This alteration does not occur if ElA-specific antibody and ETA protein are added simultaneously to the extract. In contrast, the addition of this antibody to extracts at a later time does not reverse the alteration observed in the TFIIIC binding activities. Never at any time did we note the formation of novel TFIIIC-promoter complexes after the addition of ETA to nuclear extracts. These results clearly establish that ETA mediates its effect on VAT transcription through TFIIIC in a very rapid yet indirect manner. The results also establish that a bacterially produced ETA protein can directly participate in RNA polymerase II transcription without the requirement of celiular protein synthesis or other viral proteins.
American Society for Microbiology
Transcription, E1A, Adenovirus, in vitro assay, RNA polymerase, transcription factor, TFIIIC
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