Abstract
In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.
Highlights
In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage
Single-stranded DNA fragments accumulating under DNA damage conditions serve as DNA stress signal for the SOS response and are sensed by the ATPase RecA
Based on the high structural similarity of the WYL motif-containing domain to the bacterial RNA chaperone Hfq and the known binding sites of Hfq, we identify functionally essential residues in the WYL domain of PafBC, which are likely involved in binding of a responseproducing ligand in this distinct class of transcriptional regulators
Summary
Transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. The second pathway regulates over 150 genes, including many of the LexA-controlled genes, amongst them recA, the DNA damage sensor and co-regulator of the SOS response. This other pathway operates independently of LexA and RecA, as demonstrated by deletion of recA in Mtb, which leaves upregulation of most DNA repair genes intact[9,10]. Specific interaction between PafBC and the identified DNA target regions could not be reconstituted in vitro Taken together, these results suggest that an additional “response-producing” event must take place to initiate PafBC transcription activation.
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