Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase.
Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences.
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
E A
EA
Ostrander
P
P
Benedetti
J C
JC
Wang
D000595:Amino Acid Sequence; D001483:Base Sequence; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D005656:Fungal Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013604:T-Phages / Q000201:enzymology*; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases