# Protein Dynamics in F-like Bacterial Conjugation
## 2 Dynamics of Proteins Involved in the Regulation of Bacterial Conjugation
### Traj  
The transcriptional activator of the _tra_ operon, Traj, functions through the binding of promoter P\({}_{Y}\) as a homodimer [36; 37; 38; 39]. Interaction of Traj with the promoter helps to relieve the binding of P\({}_{Y}\) DNA to histone-like nucleoid structuring proteins (H-NS) to enable binding of transcription factors [40; 41].  
Figure 2: Structures of proteins with transcription regulatory roles in the F-plasmid _tra_ operon. (**A**) The crystal structure of a truncated FinO lacking the disordered N-terminal domain (PDB ID: 1 DVO). This structure was used for modeling of small angle X-ray scattering (SAXS) data shown in (**B**), which displays the dynamic conformational ensemble involved in the binding of FinP SLII RNA (red spheres display the average structure of the best cluster of nine complexes, also shown as grey ribbons) to a truncated FinO monomer shown as a grey cartoon [28]. (**C**) The structurally conserved Per-ARNT-Sim (PAS) domain of Traj; it is the only region of a Traj structure currently solved to high resolution (PDB ID: 4 KQD). (**D**) The structure of TraR bound to RNA polymerase (RNAP) in the conformation exhibiting the highest occupancy as determined by cryo-EM. TraR, shown in blue, is seen with its N-terminal helix extending from the secondary channel of the active site in the \(\beta\)-subunit colored green. These residues are shown to interact strongly with the RNAP motif that chelates the Mg\({}^{2+}\) ion seen in grey (PDB ID: 6N57). Structural images in Figure 2A–D, Figures 4 and 5 were generated with PyMOL [35]. Figure 2B was adapted with permission from [PERSON] et al. Mapping interactions between the RNA chaperone FinO and its RNA targets. _Nucl. Acid Res._**2011**, _39_, 4450-63 [28].  
TraJ also interacts with host factor protein ArcA to aid in the transcriptional activation of the _tra_ operon by an independent mechanism [42; 43].  
Crystal structures of the N-terminal domain (NTD) of TraJ from plasmids F and pSLT have been shown to feature a Per-ARNT-Sim (PAS) fold, a common sensor motif seen in signaling proteins (Figure 2C) [41; 44; 45]. Despite dissimilar sequence homology in this domain, the structural homology of the fold is highly conserved [41]. All F-like TraJ proteins are predicted to have a PAS motif in their NTD, which has been shown to be responsible for the homodimerization of TraJ [41; 38; 46]. An NTD swapping experiment of TraJ from F-like plasmids resulted in similar levels of activation in hybrids compared to the native TraJ proteins, indicating functional interchangeability of the domain containing the PAS fold [41].  
The structure of the remaining C-terminal residues of TraJ has not been determined in high resolution; however, there is a known helix-turn-helix (HTH) motif present in all F-like TraJ homologs that has been determined to be responsible for P\({}_{Y}\) DNA recognition [41; 38; 46]. Predictably due to the HTH motif, the C-terminal domain (CTD) of TraJ displays functional specificity, as lowered activation of the _tra_ operon was seen when TraJ is interacted with the P\({}_{Y}\) from a non-cognate F-like plasmid [41; 47]. It is predicted that the dimerization of TraJ is required for its proper folding, indicating the dynamic nature of TraJ in achieving its active conformation only through dimerization [41].