A purified adenovirus 289-amino-acid E1A protein activates RNA polymerase III transcription in vitro and alters transcription factor TFIIIC.
We have previously demonstrated that a purified bacterially synthesized E1A 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 III. 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 VA1 transcription by sequestering TFIIIC. However, the addition of purified E1A to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VA1 transcription. When included in the same reaction, an E1A-specific monoclonal antibody reverses the restoration. Incubation of purified E1A 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 E1A-specific antibody and E1A 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 E1A to nuclear extracts. These results clearly establish that E1A mediates its effect on VA1 transcription through TFIIIC in a very rapid yet indirect manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Datta
S
S
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.
Soong
C J
CJ
Wang
D M
DM
Harter
M L
ML
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
D017456:Adenovirus Early Proteins; D015345:Oligonucleotide Probes; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III