Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase

Abstract

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNAmounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the β-flap domain. Like domain 4 of the promoter specificity σ factor (σ 4 ), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the β-flap domain. Nevertheless, gp33 and σ 4 are not structurally related. The gp33/β-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.| Gp33 | replication-coupled gene expression | X-ray crystallography T ranscription initiation in bacteria depends on the RNA polymerase (RNAP) catalytic core (subunit composition α 2 ββ 0 ω) and the promoter specificity subunit σ, which combine to create the RNAP holoenzyme. The σ subunit recruits RNAP to promoters through sequence-specific interactions with two conserved hexameric DNA sequence motifs, the −10 and −35 promoter elements. The −10 element is recognized by structural domain 2 of σ (σ 2 ), positioned on the clamp helices of the RNAP β′-subunit, while the −35 element is recognized by σ 4 positioned on the flap-tip-helix (FTH) of the RNAP β-subunit flap domain (1, 2). Transcription from weak promoters may be modulated by activators that typically bind to specific DNA sequences, or operators, located upstream of the −10∕ − 35 promoter elements, and stabilize the initiation complex through protein/protein interactions directly with the RNAP holoenzyme, often with the α-subunit

σ70 and PhoB activator

“…These structure models, combined with biochemical data, gave shape to the picture of the transcription machinery as we see it today. 5 Although this image is constantly being improved, [6][7][8] it still fails to depict the crucial role of the interaction of bacterial transcription factors directly with the RNAP holoenzyme upon promoter binding. Using an innovative approach, we shed some light on this particular part of the picture.…”

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

“…Some of these proteins interact directly with the host's RNAP and switch its promoter specificity to modulate the entire transcriptome in favor of phage development (Nechaev and Severinov 2003). The s 4 domain and its binding site, the b-flap domain of the RNAP core enzyme, are among the preferred targets for phage transcription regulators (Dove et al 2003;Lambert et al 2004;Nechaev et al 2004;Baxter et al 2006;Yuan et al 2009;Twist et al 2011;Osmundson et al 2012). These regulators directly bind to the interface of either s-DNA, RNAP-s, or RNAP-DNA and thus interfere with the RNAP-DNA interaction.…”

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

“…Although this approach could not obtain the full interactions between the PhoB transcription factor and the RNAP holoenzyme, they found unexpected results that the lost −35 element contacts by σ 4 domain are compensated by new ones with the PhoB activator. In another study, the authors co‐expressed the bacteriophage T4 late‐transcription coactivator gp33 and the β‐subunit flap domain of E. coli RNAP to get the gp33/β‐flap complex structure . Combining the complex structure with other biochemical and biophysical information, they successfully generated a structural model of the T4 late promoter initiation complex, which provides a structural framework for designing future experiments to functionally characterize the complex.…”

“…The strategy for preparing σ 4 chimera proteins was inspired by the work of Blanco et al 40 In this study of the PhoB transcription initiation subcomplex, the authors determined a ternary complex structure that includes the PhoB effector domain, pho box DNA and σ 4 domain fused with a shorter β-flap-tip helix. Although this approach could not obtain the full interactions between the PhoB transcription factor and the RNAP holoenzyme, they found unexpected results that the lost 42 Combining the complex structure with other biochemical and biophysical information, they successfully generated a structural model of the T4 late promoter initiation complex, which provides a structural framework for designing future experiments to functionally characterize the complex. Hence, in some cases the structural information between the transcription factor and a domain from RNAP are useful to explain the activity.…”