## 4 Structural Features of the Conserved _exo-xis_ Region Proteins
### The Off73 Genes of \(\lambda\) and Str\({}^{+}\) Phages  
Among the conserved _exo-xis_ proteins, Orf73 can be structurally modeled with high confidence since it is very similar to TraR, a bacterial protein that acts as a global regulator of stress-related genes by interacting with second regulatory channel of \(\sigma^{70}\)-dependent RNA polymerase (RNAP). TraR inhibits the expression of ribosomal RNAs and ribosomal proteins and stimulates genes associated with amino acid biosynthesis and amino acid transport without the requirement for a ppGpp cofactor that is produced during a stress response [53]. From high-resolution cryo-EM studies, TraR alters the kinetics of promoter-bound RNAP at multiple stages ultimately inhibiting or stimulating transcription from the basal level [54]. In \(\lambda\) and Str\({}^{+}\) phages, deletion of Orf73 resulted in lower survival rates upon infection and a lower efficiency of lysogenization both suggestive of a pro-lysogenic role during the phage developmental cycle [25]. Thus, as a TraR-like factor, Orf73 has the capacity to modify the expression of a wide repertoire of genes associated with the stress response through a direct interaction with RNAP [23]. Nearly all of TraR makes interactions with RNAP in an extended state [54]. The TraR structure may be considered in terms of a number of sequence hallmarks that are shared with \(\lambda\) Orf73 and the corresponding Orf73 proteins from the Str\({}^{+}\) phages \(\varphi\)24g and 933W. Of note, within the phage 933W _exo-xis_ region, there are two Orf73 sequences encoded by genes _orf73_ and _L0141_, respectively. As shown in Figure 7a, the first sequence hallmark is an acidic DxxDxA motif at the beginning of the first helix. This acidic motif makes contacts deep within RNAP. In other proteins such as DksA [55], GreA [56], and Rnk [57], the amino terminal helix is substituted by a coiled-coil that presents the acidic amino acids at a tight turn. The second sequence hallmark is a C4 zinc binding motif that is conserved among all Orf73 proteins and TraR. The third sequence hallmark is an acidic amino acid in the carboxy terminal helix [53; 58]. The fourth and final sequence hallmark ExxRK is required for interactions with the Si3 region of RNAP. Figure 7: Molecular modeling the _exo-xis_ protein, Orf73. (**a**) A sequence comparison of \(\lambda\) Orf73 and _E. coli_ TraR. Similar and identical amino acids are shaded. A dot indicates conserved cysteines that coordinate one zinc ion. (**b**) Structure of the _E. coli_ RNA polymerase (space filling representation), DNA (ribbon representation) and TraR (cartoon representation). (**c**,**d**) A ribbon and space filling representation of \(\lambda\) Orf73 following the same shading used in the sequence alignment. The AlphaFold and RoseTTAFold predicted models of \(\lambda\) Orf73 were very similar with a backbone RMSD of 0.92 A. Despite this structural similarity, each had a different accuracy associated with them (AlphaFold pLDDT = 93, RoseTTAFold pLDDT = 57). An alignment of the best representative AlphaFold \(\lambda\) Orf73 model with TraR in its RNAP bound form aligned with a C\(\alpha\) RMSD of 4.02 A due to the relative angles of the first and third \(\alpha\)-helices. The AlphaFold and RoseTTAFold models may have revealed a requirement for a conformation change upon binding. The large RMSD difference between the unbound Orf73 models and TraR made direct rigid-body docking ineffective. In lieu, a bound form of \(\lambda\) Orf73 was made directly from TraR, places coarsely into RNAP by substitution and then refined with the Rosetta 3.1 software package to minimize any steric clashes. The backbone RMSD change before and after refinement was 0.84 A indicating only minor structural adjustments were required to dock \(\lambda\) Orf73 in RNAP. The \(\lambda\) Orf73/RNAP model is presented in Figure 7b. The \(\lambda\) Orf73 model in its bound form is presented in Figure 7b,c to highlight the sequence hallmarks it shares with TraR.