Abstract
The binding of chromosomal protein HMG1 to a palindromic sequence that can form the cruciform structure in supercoiled DNA and the subsequent effect on the transcription of the sequence were examined with pBR322 and its derivative plasmids. The plasmid DNA under negative supercoiling showed a selective sensitivity to nuclease S1. HMG1 protected against the nuclease S1 digestion. The results of the filter binding assay indicated that the primary binding target of HMG1 is the single-stranded region within the cruciform in supercoiled DNA. In the transcription from pBR322 DNA in the absence of HMG1, intermediate transcripts of RNA-I, which are encoded from a DNA region containing the palindromic sequence that can form a cruciform, were accumulated with the increase in negative superhelical density whereas the full-length RNA-I was synthesized without an accumulation of intermediate transcripts in the presence of HMG1. The intermediates that accumulated in the absence of HMG1 were elongated to the final product by adding HMG1. These results suggest that the cruciform structure formed under negative supercoiling blocks transcription and that HMG1 can remove the block by altering the DNA conformation to allow the stalled RNA polymerase at the block to resume transcription.
Highlights
The binding of chromosomal protein HMGl to a palindromic sequence that can form the cruciform structure in supercoiled DNA and the subsequent effect on the transcription of the sequence were examined with pBR322 and its derivative plasmids
The intermediates that accumulated in the absence of HMGl were elongated to the final product by adding HMGl. These results suggest that the cruciform structure formed under negative supercoiling blocks transcription and that HMGl can remove the block by altering the DNA conformation to allow the stalled
The known restriction sites and the fragment sizes suggest that initial nuclease Sl digestion occurred at the cruciform originated from the palindromic sequence at residue 3065 at map position of pBR322 or a sequence in the vicinity
Summary
The binding of chromosomal protein HMGl to a palindromic sequence that can form the cruciform structure in supercoiled DNA and the subsequent effect on the transcription of the sequence were examined with pBR322 and its derivative plasmids. Javaherian et al, 1979; Makiguchi et al, 1984; Yoshida, 1987), induce superhelicity in covalently closed circular DNA (Javaherian et al, 1979; Mathis et al, 1980; Waga et al, 1989) In spite of these findings, the cellular function of the proteins is not understood several studies suggest involvement of the protein in transcription or DNA replication (Einck and Bustin, 1985). In succeeding experiments to elucidate the role of HMGl in the transcription at nuclease Sl-sensitive sites, we will show that HMGl selectively binds to the cruciform DNA under the supercoiling structure and may remove the transcriptional block caused by the cruciform formation to allow the stalled RNA polymerase to resume transcription
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