Structural basis for promoter specificity switching of RNA polymerase by a phage factor

Abstract

Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major ''-10/-35'' class promoters. Phage promoters belonging to the minor ''extended -10'' class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP b-flap domain and the C-terminal domain of the s subunit (region 4 of the s subunit [s 4 ]), thus relocating the b-flap tip and s 4 . The~45 Å displacement of s 4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

“…The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2,4,(7)(8)(9)(10)(11) that forms a part of the RNA exit channel and has been implicated in intrinsic termination and transcription pausing through direct interaction with RNA hairpins (Fig. 1) (3,(12)(13)(14).…”

Structural basis for −35 element recognition by σ4 chimera proteins and their interactions with PmrA response regulator

“…Hence, in some cases the structural information between the transcription factor and a domain from RNAP are useful to explain the activity. But in the crystal structure of the Tth RNAP holoenzyme complex with a phage factor gp33, gp33 simultaneously binds to the RNAP β‐flap tip and σ 4 domain to relocate the σ 4 domain from its binding to −35 element DNA . In this study, investigating the interactions between gp33 and β‐flap tip domain or between gp33 and σ 4 domain are not sufficient.…”

Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

“…Spx is shown to bind to a protein that comigrates with the RNAP large subunits (␤ and ␤=), and preliminary yeast two-hybrid results (S. Nakano, M. M. Nakano, and P. Zuber, unpublished data) suggest that the site of Spx interaction is the region of the ␤ subunit encompassing the flap domain previously implicated in factor interaction. Although rare, the T4 phage-encoded AsiA (38) and a recently characterized, phage-encoded transcription factor, Gp39 of Thermus thermophilus phage P23-45 (39), were found to contact both ␤ flap and region 4 of a major subunit. These additional contact sites in RNAP might explain why we found only a single monomer of Spx engaging holoenzyme despite the presence of two potential ␣ contact surfaces.…”