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, AfsR Delta TPR containing the SARP and ATPase domains and AfsR Delta C containing only the SARP domain, exl-tibited 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-(AfsR Delta C)(2)-RNAP complex was capable of initiating alpha fsS 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 alpha fsR-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 AfsRbinding site and the -10 element of the alpha fsS promoter abolished alpha fsS 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 alpha fsS 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. (C) 2007 Elsevier Ltd. All rights reserved.