LexA Represses CTXΦ Transcription by Blocking Access of the α C-terminal Domain of RNA Polymerase to Promoter DNA

Quiñones, Mariam; Kimsey, Harvey H.; Ross, Wilma; Gourse, Richard L.; Waldor, Matthew K.

doi:10.1074/jbc.m609694200

Cited by 18 publications

(23 citation statements)

References 36 publications

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“…In this study, ␦ is found to act as an activator of transcription on the rrnBP1 and abrB promoters. However, it is also possible that ␦ binds to other promoters and interacts with RNAP in a way that inhibits transcription initiation, as has been observed in the case of certain repressors (31,32). We have observed that ␦ acts as a repressor on the spo0B promoter (33), although the mechanism of repression by the protein needs to be ascertained.…”

Section: Discussionmentioning

confidence: 54%

“…Stalled Elongation Complex Assays-performed as above except that transcription initiation reactions were carried out with the abrB promoter derivatives that contains first C residues at ϩ14, ϩ24, andϩ45, respectively, and with the addition of 250 M GTP and UTP and 25 M [␣- 32 …”

Section: In Vitro Transcription Assaysmentioning

confidence: 99%

“…DNaseI Footprinting Assay-0.2 M abrB promoter DNA fragment (Ϫ95/ϩ30) labeled with 32 P at the 5Ј end of the template strand was mixed with 0, 0.5, 1.0, and 2.0 M ␦ in 50 l of transcription buffer and incubated at 37°C for 30 min. 1 l of 100 mM CaCl 2 and 0.01 unit of DNase I was added to the reaction mixtures at room temperature.…”

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

“…0.2 M of abrB promoter DNA fragment (Ϫ95/ϩ30) labeled with 32 P at the 5Ј end of the template strand was mixed with 0, 0.1, and 0.2 M FeBABE-labeled ␦ in 100 l of transcription buffer and incubated at 37°C for 15 min. Note that 0.1-0.2 M labeled ␦ was used in FeBABE footprinting assay, 10-fold less than the amount used in DNaseI footprinting to reduce the nonspecific binding of ␦ to DNA.…”

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

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

Prajapati

Sengupta

Rudra

et al. 2016

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 54%

Section: In Vitro Transcription Assaysmentioning

confidence: 99%

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

Section: Fluorescence Anisotropy Assaysmentioning

confidence: 99%

See 2 more Smart Citations

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

Prajapati

Sengupta

Rudra

et al. 2016

Journal of Biological Chemistry

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“…8 Interestingly, full repression of rstA transcription occurs by the cooperative action of a phage encoded protein, RstB, and the general repressor of the host SOS response, LexA. 9,10 Precise regulation of integrative phages replication makes sense since propagation of phage genomes during the lysogenic cycle is guaranteed by replication of the host chromosome and phage repression contributes to cell economy. Under certain conditions (generally unfavourable for the bacterial cell) the prophage is induced and re-initiates replication by its own machinery, maximizing production of phage particles.…”

Section: Introductionmentioning

confidence: 99%

CTXφ: Exploring new alternatives in host factor-mediated filamentous phage replications

2016

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For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXf, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXf and VGJf replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.

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Helicobacter pylori RNA polymerase α‐subunit C‐terminal domain shows features unique to ɛ‐proteobacteria and binds NikR/DNA complexes

2014

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Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the a subunit of RNA polymerase (aCTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although aCTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori aCTD. It is larger than other structurally determined aCTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among E-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to aCTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori aCTD to NikR:DNA complexes. We also produced aCTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori aCTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators.

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LexA Represses CTXΦ Transcription by Blocking Access of the α C-terminal Domain of RNA Polymerase to Promoter DNA

Cited by 18 publications

References 36 publications

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

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

CTXφ: Exploring new alternatives in host factor-mediated filamentous phage replications

Helicobacter pylori RNA polymerase α‐subunit C‐terminal domain shows features unique to ɛ‐proteobacteria and binds NikR/DNA complexes

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