Perspective Found: The Elusive ANTAR Transcription Antiterminator Valley Stewart1*, Herman van Tilbeurgh 2 * 1 Department of Microbiology, University of California Davis, Davis, California, United States of America, 2 IBBMC-CNRS UMR8619, Universite´ Paris-Sud, Orsay, France Regulated transcription termination provides an efficient and responsive means to control gene expression [1]. Intrinsic terminators, which consist of an RNA stem-loop followed by a poly-U tract, catalyze termination by disrupting the RNA polymerase elongation complex [2]. In antitermination, an antiterminator stem-loop is mutually exclusive with the terminator stem-loop [1,3]. In different contexts, formation of the antiterminator stem-loop is governed by a translating ribosome [1], a ligand-binding riboswitch [4,5], or a signal-responsive RNA–binding protein [3]. This latter mechanism is illuminated by Ramesh et al. [6], who studied antitermination by a broadly distributed class of signal-responsive RNA-binding proteins containing the AN- TAR domain. AmiR and NasR Define the ANTAR Regulators Studies conducted in the 1990s charac- terized two related antitermination pro- teins: AmiR, which regulates aliphatic amide catabolism in Pseudomonas aeruginosa (amiECBRS operon) [7,8], and NasR, which regulates nitrate assimilation in Klebsiella oxytoca (nasFEDCBA operon) [9,10]. RNA binding occurs through the carboxyl-terminal ANTAR domain (AmiR and NasR transcription antitermi- nation regulator) [11]. AmiR and NasR RNA binding responds to signal input mediated by the amide-binding AmiC protein [12,13] and the nitrate-binding NIT domain [14,15], respectively. AmiR and NasR target the transcribed leader RNA upstream of the amiE and nasF operons, respectively [12,14]. Both leaders encode two obvious stem-loop secondary structures: the distal intrinsic terminator (including a poly-U tract) [2], and P1, a proximal structure essential for antitermination (Figure 1A). Further anal- ysis of the nasF operon leader identified three subelements essential for NasR binding and antitermination: the P1 stem; residues A1 and G4 in the P1 loop; and part of the linker region that connects the P1 and terminator stem-loop structures [16]. However, the RNA secondary struc- ture(s) formed during antitermination re- mained a mystery. Although different ANTAR proteins use a variety of signal input domains, at least half contain a two-component signal transduction receiver domain [6,11]. Re- ceiver function is governed through phos- phorylation and dephosphorylation by a cognate signal-responsive sensor kinase [17]. For example, the ethanolamine- responsive EutW sensor kinase controls the EutV response regulator, which medi- ates antitermination control of ethanol- amine catabolism (eut operons) in Entero- coccus faecalis [18–20]. Other ANTAR proteins, including AmiR, contain a non- phosphorylated pseudo-receiver domain [13,17]. New Progress with EutV Ramesh et al. [6] now report a substantial advance toward understanding ANTAR-mediated transcription antiter- mination. The eut gene cluster contains intrinsic terminators upstream of four genes (eutP, eutG, eutS, and eutA), facilitating the identification of shared sequence features. Each of these leaders contains a terminator along with two upstream stem- loop structures, P1 and P2 [6] (Figure 1B). The eutG leader P2 structure was noted independently by Baker and Perego [20]. Similar structures are present in the amiE and nasF leaders. Strikingly, both the P1 and P2 loops contain the critical residues A1 and G4, identified as essential for NasR-mediated antitermination [16]. Moreover, the distal stem of P2 overlaps part of the terminator proximal stem [6,20], and thereby forms an antitermina- tor structure (Figure 1B). This suggests a general model for ANTAR-mediated tran- scription termination, in which the dimer- ic ANTAR protein binds simultaneously to structures P1 and P2, stabilizing the P2 antiterminator to enable transcription readthrough. (Indeed, restriction sites in- troduced into the nasF operon leader [16] destroy the P2 structure, explaining the resultant uninducible phenotype.) To test this model, Ramesh et al. first constructed a variety of site-specific alter- ations in the eutP leader, and confirmed the subelements important for EutV binding and antitermination: the P1 and P2 structures; their relative spacing; and the P1 and P2 loop residues A1 and G4 [6]. Ramesh et al. then evaluated the EutV oligomeric states, finding that the wild-type protein required phosphoryla- tion to dimerize. Truncated proteins lacking the receiver domain were isolated as dimers, although the most efficient RNA binding was observed with truncated protein that retains the central coiled-coil. This suggests that the coiled-coil enforces proper spatial orientation between the two ANTAR monomers. Presumably, each ANTAR monomer binds one of the two structures, P1 or P2. Finally, Ramesh et al. conducted bioin- formatic analyses to identify ANTAR target leaders in bacterial genomes. Ex- amples were found in a broad range of species from the Actinobacteria, Bacter- oidetes, Firmicutes, and Proteobacteria. Some of the leaders are adjacent to genes of unknown or uncertain function, where- as many others are adjacent to genes involved in inorganic nitrogen acquisition (ammonium, nitrate, or dinitrogen) or assimilation (glutamine synthetase and associated regulators). Many ethanolamine utilization clusters also were identified. Together, these results provide concrete hypotheses for the structure and function of both ANTAR domain proteins and their leader RNA targets. Moreover, the association of many ANTAR regulators Citation: Stewart V, van Tilbeurgh H (2012) Found: The Elusive ANTAR Transcription Antiterminator. PLoS Genet 8(6): e1002773. doi:10.1371/journal.pgen.1002773 Editor: William F. Burkholder, Agency for Science, Technology, and Research, Singapore Published June 7, 2012 Copyright: 2012 Stewart, van Tilbeurgh. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this article. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vjstewart@ucdavis.edu (VS); Herman.Van-Tilbeurgh@u-psud.fr (Hvt) PLoS Genetics | www.plosgenetics.org 1 June 2012 | Volume 8 | Issue 6 | e1002773