Versatility of the carboxy-terminal domain of the alpha subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA.
The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA. This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator. These results reveal a new versatility for alpha-CTD in transcriptional activation.
Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892, USA.
Bindi
B
Dangi
Angela M
AM
Gronenborn
Judah L
JL
Rosner
Robert G
RG
Martin
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024901:Drug Resistance, Multiple, Bacterial; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015534:Trans-Activators; D015533:Transcriptional Activation
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C080002:MarA protein, E coli; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit