Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

Interaction of a nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus

We have initiated a study to identify host proteins which interact with the regulatory region of the human polyomavirus JC (JCV), which is associated with the demyelinating disease, progressive multifocal leukoencephalopathy. We examined the interaction of nuclear proteins prepared from different cell lines with the JCV regulatory region by DNA binding gel retardation assays. Binding was detected with nuclear extracts prepared from human fetal glial cells, glioma cells, and HeLa cells. Little or no binding was detected with nuclear extracts prepared from human embryonic kidney cells. Competitive binding assays suggest that the nuclear factor(s) which interacted with the JCV regulatory region was different from those which interacted with the regulatory region of the closely related polyomavirus SV40. We found three areas in the JCV regulatory region protected from DNase I digestion: site A, located just upstream from the TATA sequence in the first 98-base pair (bp) repeat; site B, located upstream from the TATA sequence in the second 98-bp repeat; and site C, located just following the second 98-bp repeat. There were some differences in the ability of the nuclear factor(s) from the two brain cell lines and HeLa cells to completely protect the nucleotides within the footprint region. The results from the DNase I protective studies and competitive DNA binding studies with specific oligonucleotides, suggest that nuclear factor-1 or a nuclear factor-1-like factor is interacting with all three sites in the JCV regulatory region. In addition, the results suggest that the nuclear factor which interacts with the JCV regulatory region from human brain cell lines is different from the factor found in HeLa cells.