Summary Background Rucaparib, a poly(ADP-ribose) polymerase inhibitor, has anticancer activity in recurrent ovarian carcinoma harbouring a BRCA mutation or high percentage of genome-wide loss of heterozygosity. In this trial we assessed rucaparib versus placebo after response to second-line or later platinum-based chemotherapy in patients with high-grade, recurrent, platinum-sensitive ovarian carcinoma. Methods In this randomised, double-blind, placebo-controlled, phase 3 trial, we recruited patients from 87 hospitals and cancer centres across 11 countries. Eligible patients were aged 18 years or older, had a platinum-sensitive, high-grade serous or endometrioid ovarian, primary peritoneal, or fallopian tube carcinoma, had received at least two previous platinum-based chemotherapy regimens, had achieved complete or partial response to their last platinum-based regimen, had a cancer antigen 125 concentration of less than the upper limit of normal, had a performance status of 0–1, and had adequate organ function. Patients were ineligible if they had symptomatic or untreated central nervous system metastases, had received anticancer therapy 14 days or fewer before starting the study, or had received previous treatment with a poly(ADP-ribose) polymerase inhibitor. We randomly allocated patients 2:1 to receive oral rucaparib 600 mg twice daily or placebo in 28 day cycles using a computer-generated sequence (block size of six, stratified by homologous recombination repair gene mutation status, progression-free interval after the penultimate platinum-based regimen, and best response to the most recent platinum-based regimen). Patients, investigators, site staff, assessors, and the funder were masked to assignments. The primary outcome was investigator-assessed progression-free survival evaluated with use of an ordered step-down procedure for three nested cohorts: patients with BRCA mutations (carcinoma associated with deleterious germline or somatic BRCA mutations), patients with homologous recombination deficiencies (BRCA mutant or BRCA wild-type and high loss of heterozygosity), and the intention-to-treat population, assessed at screening and every 12 weeks thereafter. This trial is registered with ClinicalTrials.gov, number NCT01968213; enrolment is complete. Findings Between April 7, 2014, and July 19, 2016, we randomly allocated 564 patients: 375 (66%) to rucaparib and 189 (34%) to placebo. Median progression-free survival in patients with a BRCA-mutant carcinoma was 16·6 months (95% CI 13·4–22·9; 130 [35%] patients) in the rucaparib group versus 5·4 months (3·4–6·7; 66 [35%] patients) in the placebo group (hazard ratio 0·23 [95% CI 0·16–0·34]; p<0·0001). In patients with a homologous recombination deficient carcinoma (236 [63%] vs 118 [62%]), it was 13·6 months (10·9–16·2) versus 5·4 months (5·1–5·6; 0·32 [0·24–0·42]; p<0·0001). In the intention-to-treat population, it was 10·8 months (8·3–11·4) versus 5·4 months (5·3–5·5; 0·36 [0·30–0·45]; p<0·0001). Treatment-emergent adverse events of grade 3 or higher in...

Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts.

2The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing growth regimens. Before they can transcribe from specific DNA promoter sequences, bacterial core RNA polymerases (with subunit composition ␣ 2 ␤␤Ј) must combine with a dissociable sigma subunit () to form RNA polymerase holoenzyme (␣ 2 ␤␤Ј). Since the discovery of factors (6), it has become clear that these proteins are central to the function of the RNA polymerase holoenzyme. The reversible binding of alternative factors allows formation of different holoenzymes able to distinguish groups of promoters required for different cellular functions. In addition to double-strand DNA promoter recognition and binding, proteins are closely involved in promoter melting (e.g., references 31,36,49,51,74,76,128), inhibit nonspecific initiation, are targets for activators, and control early transcription through promoter clearance and release from RNA polymerase (48,49,53). Here we describe the functioning of the bacterial 54 -RNA polymerase that is the target for sophisticated signal transduction pathways (103) involving activation via remote enhancer elements (5, 95).Based on structural and functional criteria, the different factors identified in bacteria can be grouped in two classes, one of which has a single member, 54 . Many factors belong to the 70 class, the major factor which is involved in expression of most genes during exponential growth (72). 54 (also called N ) differs both in amino acid sequence and in transcription mechanism from the 70 class (80). Despite the lack of any significant sequence similarity, both types of bind the same core RNA polymerase. Nonetheless, they produce holoenzymes with different properties.With the recognition that the 54 protein represented an entirely new class of factor, what had once been regarded as an aspect of transcription restricted to higher organisms became a well-established feature of certain bacterial regulatory systems, particularly those associated with nitrogen metabolism. Activation of 54 -RNA polymerase employs specialized bacterial enhancer-binding proteins whose activating function requires nucleotide hydrolysis (94,96,122) (Fig. 1). In this system, initiation rates are controlled via regulation of the DNA melting step that is necessary for establishing the open promoter complex (85,94,97). Bacterial enhancer-dependent transcription can be studied with just two purified proteins (an activator and the 54 -RNA polymerase holoenzyme) and the appropriate DNA template, facilitating progress in understanding mechanistic aspects of 54 functioning. Below we review the biology and biochemistry of the 54 -RNA polymerase.

The bacterial phage shock protein (Psp) response functions to help cells manage the impacts of agents impairing cell membrane function. The system has relevance to biotechnology and to medicine. Originally discovered in Escherichia coli, Psp proteins and homologues are found in Gram-positive and Gram-negative bacteria, in archaea and in plants. Study of the E. coli and Yersinia enterocolitica Psp systems provides insights into how membrane-associated sensory Psp proteins might perceive membrane stress, signal to the transcription apparatus and use an ATP-hydrolysing transcription activator to produce effector proteins to overcome the stress. Progress in understanding the mechanism of signal transduction by the membrane-bound Psp proteins, regulation of the psp gene-specific transcription activator and the cell biology of the system is presented and discussed. Many features of the action of the Psp system appear to be dominated by states of self-association of the master effector, PspA, and the transcription activator, PspF, alongside a signalling pathway that displays strong conditionality in its requirement.

Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial 54 containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the 54 -holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995;Cannon et al. 1999).The activators of 54 -holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999;Cannon et al. 2000Cannon et al. , 2001. The greater part of the central domain of 54 activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The 54 protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between 54 and the activator is not well described, but an interaction with 54 can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and 54 . The binding assay was used to help define determinants in 54 and the activator needed for their interaction, and to show that binding can lead to an altered 54 -DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of 54 -dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).

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Activators of bacterial σ 54 -RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF , bound to an ATP transition state analog (ADP.AlF x ), in complex with its basal factor σ 54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF at 1.75 Å into the EM map we identify two loops involved in binding σ 54 . By comparing enhancerbinding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ 54 .Gene expression is regulated at the level of RNA polymerase (RNAP) activity. Bacterial RNAP containing the σ 54 factor requires specialized activator proteins, referred to as bacterial Enhancer-Binding Proteins (EBPs) that interact with the basal transcription complex from remote DNA sites by DNA looping (1-4). EBPs bind Upstream Activating Sequences (UAS) via their C-terminal DNA-binding domains and form higher order oligomers that use ATP-hydrolysis to activate transcription (5, 6). EBPs' central σ 54 -RNAP interacting domain is responsible for ATPase activity and transcription activation (7-9) and belongs to the larger AAA+ (ATPase Associated with various cellular Activities) family of proteins (10-12). Well studied EBPs include Phage Shock protein F (PspF), nitrogen fixation protein A (NifA), nitrogen regulation protein C (NtrC), and C 4 -dicarboxylic acid transport protein D (DctD) (1-3, 7, 13).PspF from Escherichia coli forms a stable oligomeric complex with σ 54 at the point of ATP hydrolysis (14). PspF-ADP.AlF x alters the interaction between σ 54 and promoter DNA similarly to PspF hydrolyzing ATP (15), and was thus deemed a functional hydrolysis intermediate. Activator nucleotide-hydrolysis dependent events couple the chemical energy of hydrolysis to transcriptional activation. The highly conserved and EBP-specific GAFTGA amino acid motif (Fig. S1) is a crucial mechanical determinant for the successful transfer of energy from ATP hydrolysis in EBP to the RNAP holoenzyme via σ 54 's small N-terminal * To whom correspondence should be addressed. xiaodong.zhang@imperial.ac.uk. The lack of structural information has hindered progress towards understanding the basis of this energy transfer process required for transcriptional activation. We now present a structure-function analysis of one such system using: 1) a cryo-electron microscopy reconstruction of PspF's AAA+ domain (residues 1-275, PspF ) in complex with σ 54 at the point of ATP hydrolysis (mimicked by in-situ formed ADP.AlF x ), 2) the crystal structure of apo PspF (1-275) at 1.75 Å resolution, and 3) mutational analysis. Europe PMC Funders GroupNano-electro spray mass spectroscopy of a PspF (1-275) -σ 54 complex with ADP.AlF x established that six monomers of PspF are in complex with a monomeric σ 54 , consistent with AAA+ proteins functioning as hexamers (10, 12).The 3-dimensional reconstruction of the...

Summary Transcriptional activator proteins that act upon the σ54‐containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone‐like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with σ54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the crea‐tion of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with σ54‐holoenzyme occur as a consequence of sensing the state of the γ‐phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone‐like binding activ‐ities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase‐activating proteins.