Specific transcription of an adenoviral gene that possesses no TATA sequence homology in extracts of HeLa cells.
Transcription of the adenovirus type 2 (Ad2) IVa2 gene, which contains no TATA-like sequence in the region immediately upstream of the IVa2 cap sites (Baker, C. C., and Ziff, E. B. (1981) J. Mol. Biol. 149, 189-221), has been examined in extracts of HeLa cells (Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3855-3859). Run-off transcripts of the predicted length of those initiated at the IVa2 cap sites were synthesized from different Ad2 DNA templates, each of which also contained the major late transcriptional control region. Mapping of the 5' ends of the RNA made from one template by a nuclease protection assay established the fidelity of initiation of IVa2 transcription in vitro. The efficiency of IVa2 expression in whole HeLa extracts was influenced quite dramatically by monovalent and divalent metal ion concentrations and the concentration of extract protein present in the reaction mixture. Under certain conditions, IVa2 run-off transcripts were made almost as efficiently as those from the Ad2 major late transcriptional control region. However, conditions promoting optimal IVa2 transcription in vitro did not favor recognition of the major late transcriptional control region, and vice versa: the synthesis of IVa2 and major late run-off transcripts responded differently to all parameters tested.
D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis*; D006801:Humans; D012319:RNA Polymerase II / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D012319:RNA Polymerase II; D004262:DNA Restriction Enzymes; C025651:CCCGGG-specific type II deoxyribonucleases; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific