Induction by torsional stress of an altered DNA conformation 5' upstream of the gene for a high mobility group protein from trout and specific binding to flanking sequences by the gene product HMG-T

• Published in: Biochemistry (Easton) 1988-01, Vol.27 (2), p.576-581
• Main Authors: Wright, Jonathan M, Dixon, Gordon H
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cloning, Molecular; DNA - genetics; DNA - metabolism; DNA - ultrastructure; DNA Restriction Enzymes; DNA, Superhelical - genetics; Genes; Genes, Regulator; High Mobility Group Proteins - genetics; High Mobility Group Proteins - metabolism; Molecular Sequence Data; Nucleic Acid Conformation; Nucleotide Mapping; Protein Binding; Transcription, Genetic; Trout
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00402a012

We have used enzymic and chemical probes to search for altered DNA conformations in the 5' flanking region of the gene for a high mobility group protein (HMG-T) from trout. This search was conducted in order to identify potential genetic elements that might be involved in the transcriptional control of the HMG-T gene. We identified, in supercoiled plasmid DNA molecules containing a 900 base pair insert of the 5' region of the gene, an S1-sensitive site situated within an (AT)12 sequence approximately 120 base pairs upstream from the start of the HMG-T gene. Chemical modification of supercoiled DNA with the single-strand-selective reagent bromoacetaldehyde was limited to a region coincident with the S1 nuclease site. T7 endonuclease I, a probe highly specific for four-way helical junctions, cleaved predominantly at the boundaries of the (AT)12 stretch. These data are most consistent with the interpretation that the (AT)12 sequence adopts a cruciform structure when torsionally stressed by negative supercoiling. DNase I footprinting analyses demonstrated that HMG-T protects two regions almost equidistant from the center of the (AT)12 sequence, indicating that HMG-T is a sequence-specific DNA binding protein.