Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation

Abstract

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2␣, ␤, ␤, and . However, in many Grampositive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called ␦. For over three decades since its identification, it had been thought that ␦ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that ␦ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that ␦ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of ␦. Performing biochemical and mutational analysis, we show that Bacillus subtilis ␦ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, ␦ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of ␦. Using transcription assay, we show that ␦-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that ␦ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the ؊35 promoter element for transcription activation suggests that ␦ functions as a transcriptional regulator.Transcription is the first step in gene regulation in bacteria in which RNA polymerase (RNAP) 3 together with different factors and transcriptional regulators control the gene expression. Bacterial RNAP core enzyme contains five conserved subunits: 2 ␣, ␤, ␤Ј, and . A specificity factor associates with RNAP core enzyme to form RNAP holo enzyme that is able to recognize and initiate transcription at promoters.In certain Gram-positive bacteria, including Bacillus subtilis and Staphylococcus aureus, an additional factor, called ␦, is associated with RNAP. The ␦ factor was first identified in 1975 during the purification of RNAP from phage (SP01)-infected B. subtilis (1). The protein copurified with RNAP, and therefore it was thought that ␦ functions as a subunit of RNAP. Attempts were made to characterize the functional role of the protein in transcription. Several reports suggested that ␦ was involved in promoter selection (2-5) and functioned together with A as an initiation subunit of RNAP (6, 7) or as an allosteric modulator of RNAP conformation in both initiation and the RNAP core recycling phase (5). Other reports showed that ␦ and A bind to RNAP core with negative cooperativity (8, 9), and ␦ has no effect on transcription initiation, the rate of elongation, or termination (5). Using in vitro transcription assays, several groups showed that ␦ enhances the production of transcripts from certain promoters. This increase in transcript yield in the presence of ␦ is attributed to the recycling of RNAP possibly by ␦-mediated release of RNAP from the elongation complex following transcription termination or by inhibiting the formation of s...

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“…Indeed, it has been reported that δ and σ A can bindsimultaneously to core RNAP, with negative cooperativity (51). However, other experiments indicated mutual exclusion of the binding of δ and σ A to core RNAP (43,52).…”

“…Importantly, δ has a complex effect on transcription. It inhibits initiation from weak promoters mediated by σ A (41,42), stimulates or inhibits transcription from certain other promotors (43,44) and increases RNAP recycling speed (42,43) in synergy with the DNA helicase HelD (45), probably by dissociation of stalled RNAP-DNA or RNAP-RNA complexes. Transcriptomics experiments indicate δ reduces non-specific initiation of transcription which is relatively prevalent in Gram positive bacteria (46,47).…”

“…It consists of an amino-terminal globular domain and a nucleic acid-mimicking highly acidic unstructured C-terminal half (48,49). The δ protein forms a complex with the RNAP core enzyme in a 1:1 stoichiometry (43,48,50). A truncated form of δ lacking the C-terminal half is sufficient for binding to RNAP.…”

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus

“…In support of this, those sigma factors that have increased binding to RNAP upon deletion ( S in E. coli [24], B / F in cyanobacteria [25], and B in S. aureus) are all major components of the general stress response in their respective organism (45)(46)(47)(48)(49). Furthermore, as with factors, the release of ␦ from RNAP has the potential for profound alterations in gene expression, as a recent publication demonstrates the ability of ␦ to selectively affect individual promoters (7,8) via binding to specific promoter elements (50). As the disruption of rpoE results in slower bacterial growth in a variety of organisms (e.g., an extended lag phase [8,51,52]), removal of this subunit from RNAP may actually present a survival advantage during stress, redirecting resources away from division and toward repair and cellular maintenance.…”

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“…Moreover, we have included the RNA polymerase-interacting protein HelD and the nonessential delta subunit (RpoE). HelD binding stimulates transcription in an RpoE-dependent manner, suggesting that these two accessory proteins are important to allow rapid growth (59,60). Interestingly, both proteins are absent from the rather slowly growing M. mycoides strain JCVI-syn3.0.…”