An artificial activator that contacts a normally occluded surface of the RNA polymerase holoenzyme.
Many activators of transcription are sequence-specific DNA-binding proteins that stimulate transcription initiation through interaction with RNA polymerase (RNAP). Such activators can be constructed artificially by fusing a DNA-binding protein to a protein domain that can interact with an accessible surface of RNAP. In these cases, the artificial activator is directed to a target promoter bearing a recognition site for the DNA-binding protein. Here we describe an artificial activator that functions by contacting a normally occluded surface of promoter-bound RNAP holoenzyme. This artificial activator consists of a DNA-binding protein fused to the bacteriophage T4-encoded transcription regulator AsiA. On its own, AsiA inhibits transcription by Escherichia coli RNAP because it remodels the holoenzyme, disrupting an intersubunit interaction that is required for recognition of the major class of bacterial promoters. However, when tethered to the DNA via a DNA-binding protein, AsiA can exert a strong stimulatory effect on transcription by disrupting the same intersubunit interaction, contacting an otherwise occluded surface of the holoenzyme. We show that mutations that affect the intersubunit interaction targeted by AsiA modulate the stimulatory effect of this artificial activator. Our results thus demonstrate that changes in the accessibility of a normally occluded surface of the RNAP holoenzyme can modulate the activity of a gene-specific regulator of transcription.
Gregory
Brian D
BD
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA.
Deighan
Padraig
P
Hochschild
Ann
A
D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
C078876:AsiA protein, Enterobacteria phage T4; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases