Structural and Molecular Insights into the Transcription Activation Mechanism of the WYL Domain-Containing Activator, DriD

Abstract

During a DNA damage event, bacteria maintain genomic integrity via the canonical SOS response, which is activated upon sensing DNA damage signals, leading to inhibition of bacterial cell division and a subsequent increase in expression of DNA damage response genes. Proper feedback between cell division and DNA damage repair is a hallmark of the SOS response, ensuring damaged genomic material is not passed on to daughter cells. However, recent studies in multiple bacterial species, including Caulobacter crescentus (C. crescentus), show evidence of alternative DNA damage response pathways, independent of the major SOS response. In C. crescentus, an alternative DNA damage response pathway was recently shown to be activated by the novel transcription activator, DriD, which regulates the transcription of multiple DNA damage and cell cycle control genes. DriD is a member of the newly discovered class of WYL Transcription Factors (TFs) which in addition to SOS independent DNA damage repair, play critical roles in a variety of bacterial processes such as phage defense, stress response, and CRISPR Cas adaptive immunity. DriD responds to DNA damage by recognition of the genotoxic signal, ssDNA, which accumulates during double stranded DNA breaks and binds the WYL domain of DriD. Upon binding ssDNA, DriD is activated to make base specific contacts with the double stranded cognate DNA and induce transcription of DriD regulated genes. The full-length crystal structure of DriD bound to effector and cognate DNA was recently solved in our laboratory (PDB code: 8TP8). This structure revealed DriD is a 327-residue homodimer containing an N-terminal winged helix-turn-helix (wHTH) motif, a linker, a three-helix bundle (3HB), a central WYL-domain (Trp-Tyr-Leu motif), and a WYL C-terminal extension (WCX) dimerization domain. The structure reveals how ssDNA is bound in the WYL domain and how DriD anchors onto cognate DNA. However, the mechanism by which ssDNA binding allosterically transduces the signal to permit base specific contacts between DriD and the cognate DNA remained unclear. Also unclear is how DriD forms a complex with RNA polymerase to activate transcription from different promoter sites. In this dissertation, I describe the first biochemical investigation into the full mechanism of transcription activation by a WYL TF. This dissertation explores various aspects of DriD including its cognate DNA recognition mechanism, allostery upon ssDNA recognition, and how DriD coordinates with the RNA polymerase holoenzyme. Through fluorescence polarization studies, I find DriD utilizes two highly conserved arginine residues to read guanine bases in the cognate DNA sequence. Using X-ray crystallography, I discovered the three-helix bundle of a truncated version of DriD without the wHTH DNA binding domain is highly disordered in its apo state. A collaborative study revealed the wHTH DNA binding domain contacts and shields the three-helix bundle, thus exposing an autoinhibitory mechanism between the wHTH DNABDs and the 3HB. I also describe cryogenic electron microscopy structures of DriD in complex with RNA polymerase holoenzyme at multiple promoter sites. As DriD homologs are found in a variety of pathogenic species of bacteria, these studies will illuminate the mechanism employed by DriD and other homologous WYL TF activators involved in a variety of bacterial processes. This research aims to understand a new class of transcription activators that aid in bacterial survival in damaging environments. A thorough understanding of DriD, a representative member of WYL TF activators, may set the foundation for the discovery of a novel class of antibiotics.