Transcription is the first step in gene regulation and is performed by RNA polymerase (RNAP). RNAP core enzyme that catalyzes the RNA synthesis contains five conserved subunits: two α, one each of β, β’, and ω. To initiate transcription from a promoter DNA, the RNAP core must bind to the σ subunit to form the RNAP holoenzyme. In some promoters, RNAP requires the help of transcriptional regulator(s) to activate transcription. The transcriptional regulators often interact with the α CTD (C-terminal domain of the α subunit of RNAP) that is connected to its N-terminal domain via a flexible linker.1, 2, 3, 4, 5, 6, 7, 8, 9 Earlier studies reveal that α CTD has three distinct domains: (I) the 287 determinant (residues 285–290, 315, 317, 318) responsible for interaction with the regulator; (ii) the 265 determinant (residues 265, 294, 296, 298, 299, 302) responsible for interaction with the DNA UP element, and (iii) the 261 determinant (residues 257, 258, 259, and 261) responsible for interaction with the σ region 4 (σ R4).1, 10, 11, 12, 13 However, there are significant pieces of evidence about the versatility of these determinants in their interaction with the regulators.14, 15, 16, 17, 18, 19 For example, the 265 determinant of α CTD is involved in its interaction with the transcription regulator MarA and SoxS.14, 15, 16 For Spx, a completely new set of residues (K267, N264, R268, R261, L258, L256, and L258) from B. subtilis α CTD is involved in the interaction.19 In the classical model of transcription activation at Class I promoters of E. coli, the transcriptional regulator, CAP (catabolite activator protein) binds to the DNA centred at −61.5 and interacts with α CTD through the ‘287 determinant’.1, 20, 21 The α CTD that remains sandwiched between CAP and RNAP also interacts with the DNA site through the ‘265 determinant’ and with the σ R4 through the ‘261 determinant’ (Figure 1A). In transcription activation at Class II promoters, CAP binds to DNA centred at −41.5 and adjacent to the RNAP binding site. The αCTD interacts with CAP at the opposite face of RNAP through the 287 determinant and contacts the nearby DNA site through the 265 determinant.11, 22, 23, 24, 25 Here, CAP also interacts with the α NTD as well as σ R4 (Figure 1B).12, 13 There are many other regulators in which the transcription activation closely resembles the above models of gene regulation.6, 7, 8, 9, 14 Recently another class of transcription activation has been proposed in which the two α CTDs interact with each other acting as a bridge between the transcription regulator SoxS and σ R4. Here one of the α CTDs interacts with SoxS, bound to DNA, through the 265 determinant and the other α CTD interacts with both the UP element through the 265 determinant and σ R4 through the 261 determinant (Figure 1C).16 In all the classes of promoter activation, the interactions of transcription regulator with α CTDs lead to the stabilization of the transcriptional machinery at the promoter through a ‘simple recruitment mechanism’.12 The global regulator CAP is present in E. coli, but not in B. subtilis. Incidentally, Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes and other firmicutes contain the global transcriptional modulator δ factor, which is not present in any gram-negative bacteria including E. coli.26

δ was first identified four decades ago during purification of RNAP from B subtilis.26 Since then a lot of functions of δ have been proposed, but the way δ functions remain ambiguous.27, 28, 29, 30, 31, 32, 33, 34, 35 The most acceptable function of δ is the recycling of RNAP from the stalled elongation complex and thereby efficient recycling of RNAP during transcription events.30, 34 Consistent with the idea that δ is associated with the RNAP core, the structure of RNAP-δ-HelD revealed that the binding of the N terminal domain of δ (δ NTD) at the β’ subunit of RNAP and probable binding location of its C terminal region (δ CTR) inside the active centre cleft.36, 37 Since δ CTR is highly negatively charged and can compete with RNA, the binding of δ to the RNAP core may explain the possible mechanism of release of RNA from the elongation complex.36 The binding of δ to the β’ subunit of RNAP was also observed in the actively transcribing and translating expressome from M. pneumoniae.38 However, the ability of δ to bind the RNAP core, but not to RNAP holo and its ability to function in concert with the holoenzyme makes its mechanism of function ambiguous.39 Removal of the rpoE gene (encoding δ) does not exhibit any major effect in vivo except a prolonged lag phase during growth and an altered cell morphology in B. subtilis.35 The lack of δ impacts on the competitive fitness of B. subtilis cell,40 pathophysiology in S. pyogenes41 and causes defects in biofilm formation and adaptation under stress conditions in S. mutans.42 Most significantly in many of the pathogens, the rpoE gene is involved in the upregulation and downregulation of many genes including the genes encoding virulence factors.43 This indicates that gene regulation by δ is promoter-specific. Thus, although δ is involved in the recycling of RNAP, this cannot be the sole reason for the δ dependent transcriptional modulation. The protein may also function as a transcriptional regulator.

In our previous studies, we have shown that δ could act both as a transcriptional activator or a repressor depending on its DNA binding sites (A-rich sequence) at the promoter.39, 44 If the δ binding site is located within the promoter region mostly at the −35 element of the extended −10 promoters, δ behaves as a repressor. On the other hand, when δ binds at the A-rich sequence immediately upstream of the −35 element, δ acts as an activator. δ activates transcription both by facilitating the open complex formation and recycling of polymerase. Mutation at the δ-binding site at DNA abrogates the activation of transcription. These studies explained the possible mechanism of the up- and down-regulation of genes by δ. However, the way δ interacts with RNAP in the context of RNAP-δ-DNA ternary complex to activate transcription still remains unknown. In this study, we show that δ binds to the same promoter DNA fragment with 10 times higher affinity when RNAP is present at the promoter. When RNAP escapes the promoter upon transcription elongation, δ loses its affinity to its binding site at the promoter. We further show that, contrary to the conventional transcriptional activators mediated recruitment of RNAP at the promoter, RNAP stabilizes δ at the promoter through an interaction with its α CTD domain. Two side-by-side amino acid residues R293 and K294 of α CTD (located at the 265 determinant) are responsible for the interaction with δ. A point mutation on any of these two residues abolishes the δ dependent enhancement of the open complex formation and transcription activation. Most strikingly, interaction with the residue R293 stabilizes δ, whereas, interaction with K294 facilitates the open complex formation. Based on our experimental data we propose a model of transcription activation in which RNAP recruits δ at the −41 site of upstream promoter DNA, and the protein, in turn, facilitates the open complex formation. Thus, the proposed model is similar to the transcription activation at class II as the transcription factor binds to the −41 site of DNA and interacts with the 265 determinant of α CTD, but distinct from the model as the transcription factor is not involved in the recruitment of RNAP at the promoter, but enhances the formation of the open complex by RNAP.