A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

Abstract

A simple approach has been developed for the unambiguous identification and purification of sequencespecific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44-to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.The rate of transcription initiation is an important determinant of gene expression in eucaryotic organisms. Considerable effort has been devoted to elucidating the molecular mechanisms responsible for regulating the transcription initiation rate of specific genes. Genetic analysis of RNA polymerase II transcription units has defined at least three qualitatively different types of cis-acting DNA regulatory elements: the TATA box, upstream promoter elements, and transcriptional enhancers. Recently, the gel electrophoresis DNA-binding assay and the DNase footprinting assay have been used to demonstrate that each of these types of elements interacts with one or more sequence-specific DNAbinding proteins (4,6,21,23). Furthermore, proteins which bind to the TATA box and to at least three distinct upstream promoter elements have been shown to stimulate transcription in vitro by RNA polymerase II (4,5,6,21,22,32). One of these transcription factors, MLTF or USF, binds to and stimulates transcription from the major late promoter (MLP) of adenovirus (4,18,32). An understanding of the mechanisms by which MLTF and other transcription factors control transcription initiation requires the identification, purification, and characterization of these proteins. In addition, isolation of the genes encoding these proteins will permit study of their regulation during the course of cell growth and development and will allow genetic analysis of the structural characteristics which specify their transcriptional interactions.The existence of MLTF was first suggested by the dependence in vivo of transcription from the adenovirus type 2 MLP on sequences located 50 to 66 base pairs (bp) upstream of the transcription...

Complete DNA sequence of canine adenovirus type 1.

“…An AT-rich region upstream of the left ITR shows some similarity to the packaging signals identified in Ad2 (Gra$ ble & Hearing, 1990). A sequence with a 10\12 match to the major late transcription factor (MLTF\ USF) binding site (Carthew et al, 1985 ;Chodosh et al, 1986) is located between bases 7398 and 7409. The presence of other transcriptional elements (CCAAT box, AP-1 site and TATA box) in this region suggests that the major late promoter (MLP) is located here, although this is not the same position as that observed in human adenoviruses (approximately 5n5 kb, within the pol gene).…”

The 30‐kDa rat liver transcription factor nuclear factor 1 binds the rat growth‐hormone proximal silencer

“…A crude nuclear extract was prepared from adult rat liver [24,251 and further enriched on a heparin-Sepharose (HS) column (Pharmacia) as previously described [26]. Fractions (designated as HS followed by the fraction number) were eluted with a linear 0.1-1.0 M KC1 gradient, dialyzed against buffer A (50 mM KC1,4 mM MgCL 20 mM K3P04, pH 7.4, 1 mM 2-mercaptoethanol, 20% glycerol) for 1 h at 4"C, and individually tested in a gel mobility shift assay to define which of these supports an rGH silencer-1 -binding activity.…”

Interaction of upstream stimulatory factor with the human heme oxygenase gene promoter

“…The protein fraction was subsequently applied to a heparin-agarose column equilibrated with buffer A containing 0.1 M KCl, followed by extensive washing with buffer A containing 0.2 M KC1. The proteins bound to the heparin-agarose were then eluted with buffer A containing 0.3 M KC1 (USF fraction) [17] and used for cell-free transcription as well as for binding assays.…”

“…major late transcription factor [17] originally identified in HeLa cells. We have therefore provided several lines of evidence that heme oxygenase transcription factor is a rat homolog of USF [14].…”