## 5 Non-Conserved _exo-xis_ Region Proteins of Interest  
### Bacteriophage \(\lambda\) Orf55 Is a Possible Modulator of RNA Polymerase  
The final gene in the _exo-xis_ region of \(\lambda\) bacteriophage is an uncharacterized ORF simply called _orf55_. There is no _orf55_ homolog in the Stx\({}^{+}\) phage \(\varphi\)24g; however, in phage 933W, there a portion of _orf55_ at an analogous location near the _xis_ gene. AlphaFold and RoseTTAFold both predicted a similar fold for Orf55 consisting of three \(\beta\)-strands and one \(\alpha\)-helix (0.94 A RMSD). A search against the PDB revealed three homologous structures: The first candidate was p56 (PDB: 4L5N), a uracil glycosidase inhibitor from _B. subtilis_ phage \(\varphi\)29 [59]. While structurally similar (2.8 A RMSD), it was only 5% identical at the sequence level. The second candidate (PDB: 4 NJC; 2.1 A RMSD) was the \(\varepsilon\) subunit of RNAP of gram-positive bacteria encoded by the gene, _rpoY_[60]. The \(\varepsilon\) subunit had no sequence identity with Orf55. The third and final candidate was Gr2 (PDB: 4 LLG), an inhibitor of RNAP from phage T7 with an RMSD of 2.07 A and an overall sequence similarity of 20% [61].  
The T7 phage Gr2 protein interacts with \(\beta^{\prime}\) subunit of RNAP and translocates a domain (1.1) of \(\sigma^{70}\) to block the downstream DNA duplex channel and effectively shut down host transcription in favor of transcription by T7 polymerase. To examine how Orf55 may bind RNAP in an analogous way to T7 phage Gr2 protein, a molecular model was made by substituting Orf55 for Gr2 in the Gr2/RNAP complex and then using the Rosetta software suite to make minor backbone moves to minimize steric clashes and to maximize favorable contacts. The results of this modeling study are presented in Figure 8.  
In the Orf55/RNAP model, Orf55 is positioned between \(\beta^{\prime}\) and \(\sigma^{70}\)\({}_{1.1}\) and makes extensive contacts with both proteins. It is known that substitution of one of two arginines to alanine near the third \(\beta\)-strand of Gr2 are deleterious both in terms of RNAP inhibition (\(\sim\)20% of wild type) and affinity (\(\sim\)12% of wild type). The analogous amino acids in the Orf55 model are N43 and R45; thus, one amino is substituted and one is identical. Of the two positions, N43 is partially surface exposed and consequently, not necessarily incompatible with the complex. The second position, R45, is positioned deeper at the interface with the \(\beta^{\prime}\) subunit. Favorable ionic contacts in the model are observed with E47 of Orf55 and E1158 of the \(\beta^{\prime}\) subunit. At the other interface, a cluster of favorable hydrophobic contacts in the model are made between W9 of Orf55 and L9, I44, and I48 of While this model is speculative, the presence of Orf55 during early stages of infection may serve an antagonistic role against \(\sigma^{70}\)-mediated gene expression, assuming its affinity for RNAP is moderate. Expression of genes related to the stress response would remain unaffected. Remodeling the dynamics of gene expression, even transiently, could have far reaching effects in the bacterial host and the efficiency at which \(\lambda\) proceeds through lytic developmental pathway.