Protein factor(s) binding independently to two different regions of the adenovirus 2 major late promoter.

The protein factor(s) in a fraction from the HeLa cell nuclear extract required for specific in vitro transcription can specifically bind to adenovirus 2 major late promoter (Ad 2 MLP) DNA. We demonstrate by in vitro footprinting assay that there are two asymmetric protected regions covering the TATA box and the nucleotides upstream from the TATA box. In the coding strand, the DNAse I protected regions span from nucleotides -10 to -50 and from -52 to -68. In the noncoding strand, the protected regions span from nucleotides -10 to -32 and from -45 to -65. Using different Ad 2 MLP point mutants in this assay, we show that the transcriptional down mutants of the TATA box (AC-30 and AC-28) abolish the binding of protein factor(s) to the TATA box but do not affect binding in the upstream region. The new upstream transcriptional down mutant (TA-56) abolishes the binding of protein factor(s) in the upstream region but does not affect binding to the TATA box. The mutants which do not affect transcription efficiency (GA-51 and CG-61) do not modify the binding to either the TATA box or the upstream region. Methylation protection experiments show that the guanosines at -58 and -60 in the coding strand and at -57 (probably also -55) in the noncoding strand are in close contact with protein factor(s). The results indicate that the TATA box and its upstream region of Ad 2 MLP are independently bound by at least two different factors in vitro.