AfsR recruits RNA polymerase to the afsS promoter: a model for transcriptional activation by SARPs.
AfsR, a protein belonging to the Streptomyces antibiotic regulatory protein (SARP) family, is a global regulator of secondary metabolism in Streptomyces coelicolor A3(2). AfsR consists of three major functional domains: an N-terminal SARP domain, a central ATPase domain, and a C-terminal tetratrico peptide repeat (TPR) domain. Two truncated AfsR proteins, AfsRDeltaTPR containing the SARP and ATPase domains and AfsRDeltaC containing only the SARP domain, exhibited the same DNA-binding specificity as that of full-length AfsR. Two monomers bound cooperatively to a direct repeat located eight nucleotides 5' to the -10 element of the afsS promoter. Both truncated AfsR proteins, as well as full-length AfsR, were able to form ternary complexes with the afsS promoter and RNA polymerase (RNAP), although RNAP alone could not bind to the DNA. The DNA-(AfsRDeltaC)(2)-RNAP complex was capable of initiating afsS transcription in vitro, indicating that the ATPase and TPR domains are dispensable for the basic function of AfsR as a transcriptional activator. However, the ATPase domain was required to fully compensate for the defect in actinorhodin production in an afsR-disrupted mutant, suggesting that the ATPase domain exerts a regulatory function on the basic SARP domain. Deletion or addition of even a single nucleotide between the AfsR-binding site and the -10 element of the afsS promoter abolished afsS transcription both in vitro and in vivo, indicating that the recruitment of RNAP by AfsR to the correct location relative to the -10 element is critical for transcriptional activation. Since SARP-binding sites with similar direct repeats are located at the same position relative to the -10 element of their target promoters as is the afsS binding site, the SARP family members presumably activate transcription of their targets by recruiting RNAP to the promoter, where a ternary DNA-SARP-RNAP complex competent for transcriptional initiation is formed.
Tanaka
Akiko
A
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Takano
Yuji
Y
Ohnishi
Yasuo
Y
Horinouchi
Sueharu
S
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C066581:AfsR protein, Streptomyces coelicolor; D012321:DNA-Directed RNA Polymerases