BglG, the transcriptional antiterminator of the bgl system, interacts with the β′ subunit of the Escherichia coli RNA polymerase

Abstract

The Escherichia coli BglG protein antiterminates transcription at two terminator sites within the bgl operon in response to the presence of ␤-glucosides in the growth medium. BglG was previously shown to be an RNAbinding protein that recognizes a specific sequence located just upstream of each of the terminators and partially overlapping with them. We show here that BglG also binds to the E. coli RNA polymerase, both in vivo and in vitro. By using several techniques, we identified the ␤ subunit of RNA polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the Nterminal region of ␤. The ␤ subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase ␤ subunit are discussed.The bgl operon in Escherichia coli, induced by ␤-glucosides, is regulated by two of its gene products, BglG, a transcriptional regulator, and BglF, a membrane-bound sensor (1). Transcription from the bgl promoter initiates constitutively, but in the absence of inducer, most transcripts terminate prematurely at one of two -independent terminators within the operon; in the presence of inducer, BglG prevents termination at these sites (2, 3). The mechanism by which BglG antiterminates transcription differs from the antitermination mechanisms which operate in and the mechanisms of attenuation at amino acid biosynthetic operons, as well as at the pyrB1 and ampC operons (4). BglG is an RNA-binding protein that recognizes and binds to a specific sequence on the bgl transcript, which partially overlaps with each of the bgl terminators (5). BglF, the ␤-glucosides phosphotransferase, regulates the activity of BglG by reversible phosphorylation (6-9), which modulates BglG dimeric state (10). The phosphorylation and dimerization sites on BglG were recently mapped (11-13).Systems that resemble the bgl system were identified in different organisms (14). The most studied are the two sac systems in Bacillus subtilis, which regulate transcription of sacB and sacPA according to sucrose availability. Expression of these loci is regulated at the level of transcription antitermination by the two BglG homologues, SacY and SacT,. Similarly to BglG, SacY is reversibly phosphorylated in vivo (18). The RNA sequences recognized by SacY and SacT closely resemble the target site for BglG in the bgl transcript (19, 16) and also have the potential to fold into a stem-loop structure that partially overlaps with the respective terminators (5, 20). † It was suggested that the antiterminators of the BglG family block the formation of the terminator structures by stabilizing an alternative RNA conformation. The question then arises as to whether interaction with the nascent RNA chain is sufficient for implementing antitermination or additional interactions with the transcription machinery are required. In this paper we provide both in vivo and in vitro evidence for the interaction of BglG with the ␤Ј subunit of E. coli RNA poly...

PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

“…Purified maltose binding protein (MBP) (NEB) was loaded as a positive control. Gels were subjected to Western blot analysis essentially as described previously (29). Incubation with the primary antibody (anti-MBP, monoclonal, diluted 1:3,000) was carried out for 1 h at room temperature (RT), and incubation with the secondary antibodies (goat anti-mouse, diluted 1:20,000) was for 30 min at RT.…”

Analysis of cis- and trans- Acting Factors Involved in Regulation of the Streptococcus mutans Fructanase Gene ( fruA )

“…Substrate induction by transcriptional antitermination, in which the antiterminator binds to the upstream region of the RNA hairpin stem of the intrinsic terminator transcript, protecting the elongation complex from destabilization (43), is well documented in gram-positive bacteria (23,27,43). Previously, we identified in the 5Ј UTR of the fruA transcript two overlapping stem-loops, designated SL1 at positions 80 to 106 (relative to the transcription initiation site [TIS]) and SL2 at positions 94 to 116, with the potential to function as transcriptional terminators (Fig.…”

The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation

“…Transcription from the bgl promoter initiates constitutively, but in the absence of ␤-glucosides, most transcripts terminate prematurely at one of two -independent terminators within the operon; in the presence of an inducer, BglG allows transcription through these sites by binding to the bgl transcript (3)(4)(5). BglG also recognizes and interacts with the ␤Ј subunit of E. coli RNA polymerase (6). BglF regulates BglG activity by reversibly phosphorylating it depending on ␤-glucoside availability (7)(8)(9), thus modulating its dimeric state (10).…”