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.
Regulated transcription termination provides an efficient and responsive means to control gene expression . Intrinsic terminators, which consist of an RNA stem-loop followed by a poly-U tract, catalyze termination by disrupting the RNA polymerase elongation complex . In antitermination, an antiterminator stem-loop is mutually exclusive with the terminator stem-loop , . In different contexts, formation of the antiterminator stem-loop is governed by a translating ribosome , a ligand-binding riboswitch , , or a signal-responsive RNA–binding protein . This latter mechanism is illuminated by Ramesh et al. , who studied antitermination by a broadly distributed class of signal-responsive RNA-binding proteins containing the ANTAR domain.
AmiR and NasR Define the ANTAR Regulators
Studies conducted in the 1990s characterized two related antitermination proteins: AmiR, which regulates aliphatic amide catabolism in Pseudomonas aeruginosa (amiECBRS operon) , , and NasR, which regulates nitrate assimilation in Klebsiella oxytoca (nasFEDCBA operon) , . RNA binding occurs through the carboxyl-terminal ANTAR domain (AmiR and NasR transcription antitermination regulator) . AmiR and NasR RNA binding responds to signal input mediated by the amide-binding AmiC protein ,  and the nitrate-binding NIT domain , , respectively.
AmiR and NasR target the transcribed leader RNA upstream of the amiE and nasF operons, respectively , . Both leaders encode two obvious stem-loop secondary structures: the distal intrinsic terminator (including a poly-U tract) , and P1, a proximal structure essential for antitermination (Figure 1A). Further analysis 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 . However, the RNA secondary structure(s) formed during antitermination remained a mystery.
Figure 1. RNA-binding proteins stabilize antiterminator structures.
Alternative stem-loop structures in transcribed leader regions from representative operons are shown. Terminator stem regions that participate in alternative structures are in blue, whereas antiterminator regions are in red. (A) NasR-responsive nasF operon leader in termination conformation. Dots indicate the critical residues A1 and G4 in the P1 and P2 loops. (B) Each ANTAR monomer is hypothesized to bind one of the loops, P1 and P2. Stabilizing the P2 antiterminator structure permits transcription readthrough  by shortening the terminator stem and separating it from the poly-U tract . (C) LicT-responsive bglP operon leader in termination conformation. RAT is the ribonucleic antiterminator. (D) Each CAT (co-antiterminator) monomer binds to the antiterminator stem. Unusual base-pairing within the antiterminator stem is depicted schematically . Stablilizing the antiterminator structure permits transcription readthrough  by shortening the terminator stem and separating it from the poly-U tract.doi:10.1371/journal.pgen.1002773.g001
Although different ANTAR proteins use a variety of signal input domains, at least half contain a two-component signal transduction receiver domain , . Receiver function is governed through phosphorylation and dephosphorylation by a cognate signal-responsive sensor kinase . For example, the ethanolamine-responsive EutW sensor kinase controls the EutV response regulator, which mediates antitermination control of ethanolamine catabolism (eut operons) in Enterococcus faecalis –. Other ANTAR proteins, including AmiR, contain a non-phosphorylated pseudo-receiver domain , .
New Progress with EutV
Ramesh et al.  now report a substantial advance toward understanding ANTAR-mediated transcription antitermination. 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  (Figure 1B). The eutG leader P2 structure was noted independently by Baker and Perego . 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 . Moreover, the distal stem of P2 overlaps part of the terminator proximal stem , , and thereby forms an antiterminator structure (Figure 1B). This suggests a general model for ANTAR-mediated transcription termination, in which the dimeric ANTAR protein binds simultaneously to structures P1 and P2, stabilizing the P2 antiterminator to enable transcription readthrough. (Indeed, restriction sites introduced into the nasF operon leader  destroy the P2 structure, explaining the resultant uninducible phenotype.)
To test this model, Ramesh et al. first constructed a variety of site-specific alterations 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 . Ramesh et al. then evaluated the EutV oligomeric states, finding that the wild-type protein required phosphorylation 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 bioinformatic analyses to identify ANTAR target leaders in bacterial genomes. Examples were found in a broad range of species from the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Some of the leaders are adjacent to genes of unknown or uncertain function, whereas 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 with aspects of nitrogen metabolism provokes intriguing questions about their physiology and evolution.
Comparison to CAT Domain Antitermination
Proteins of the BglG-LicT-SacY family, present in both gram-negative and gram-positive lineages, control genes for sugar catabolism. These homodimeric proteins contain PRD domains (PTS regulation domain) that place them under control of the phosphotransferase system for sugar uptake . The carboxyl-terminal CAT (co-antiterminator) domain interacts with the RAT (ribonucleic antiterminator) element in the transcribed leader region. The CAT-stabilized antiterminator is mutually exclusive with the terminator structure  (Figure 1C and 1D).
The CAT domain is a homodimer of four-stranded β-sheets . Phosphorylation results in massive structural changes  that bring the two CAT monomers into proper alignment to interact with a distorted minor grove in the antiterminator hairpin stem . This stem is interrupted by two non-identical asymmetric internal loops, which are recognized similarly by the protein dimer. Sequence analysis suggests that this RNA-binding mode likely is conserved in homologous systems .
How Does ANTAR Bind RNA?
X-ray structures are known for three ANTAR domain proteins, each with a different amino-terminal input domain: AmiR, pseudo-receiver ; Mycobacterium tuberculosis Rv1626, receiver ; and NasR, NIT . Each protein contains an α-helical coiled-coil connecting the two domains, but each structure reveals a different spatial orientation of the ANTAR monomers and a different configuration of the coiled-coil. It is likely that signal is propagated through the coiled-coil to bring the ANTAR monomers into proper alignment for RNA binding .
The ANTAR domain adopts a helix-turn-helix conformation, but how it binds RNA is unknown. RNA-binding helix-turn-helix proteins include σ70 region 4.2, which binds the −35 region of promoter DNA as well as 6S RNA , and Ffh, which binds 4.5 S RNA in the signal recognition particle . Both of these RNA targets form bulged stem-loop structures, so the binding sites have characteristics of dsRNA.
Future progress in understanding ANTAR-mediated antitermination requires knowledge of ANTAR domain conformation and interaction with its RNA target; conformational shifts mediated through the coiled-coil; and kinetics of RNA binding in relation to transcription-driven formation of the P1, P2, and terminator structures.
- 1. Yanofsky C (2007) RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria. RNA 13: 1141–1154.
- 2. Peters JM, Vangeloff AD, Landick R (2011) Bacterial transcription terminators: the RNA 3′-end chronicles. J Mol Biol 412: 793–813.
- 3. Henkin TM, Yanofsky C (2002) Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions. Bio Essays 24: 700–707.
- 4. Roth A, Breaker RR (2009) The structural and functional diversity of metabolite-binding riboswitches. Annu Rev Biochem 78: 305–334.
- 5. Gutiérrez-Preciado A, Henkin TM, Grundy FJ, Yanofsky C, Merino E (2009) Biochemical features and functional implications of the RNA-based T-box regulatory mechanism. Microbiol Mol Biol Rev 73: 36–61.
- 6. Ramesh A, DebRoy S, Goodson JR, Fox KA, Faz H, et al. (2012) The mechanism for RNA recognition by ANTAR regulators of gene expression. PLoS Genet 8: e1002666. doi:10.1371/journal.pgen.1002666.
- 7. Drew R, Lowe N (1989) Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism. J Gen Microbiol 135: 817–823.
- 8. Wilson SA, Wachira SJM, Norman RA, Pearl LH, Drew RE (1996) Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon. EMBO J 15: 5907–5916.
- 9. Goldman BS, Lin JT, Stewart V (1994) Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al. J Bacteriol 176: 5077–5085.
- 10. Lin JT, Stewart V (1996) Nitrate and nitrite-mediated transcription antitermination control of nasF (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al. J Mol Biol 256: 423–435.
- 11. Shu CJ, Zhulin IB (2002) ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins. Trends Biochem Sci 27: 3–5.
- 12. Norman RA, Poh CL, Pearl LH, O'Hara BP, Drew RE (2000) Steric hindrance regulation of the Pseudomonas aeruginosa amidase operon. J Biol Chem 275: 30660–30667.
- 13. O'Hara BP, Norman RA, Wan PT, Roe SM, Barrett TE, et al. (1999) Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex. EMBO J 18: 5175–5186.
- 14. Chai W, Stewart V (1998) NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro. J Mol Biol 283: 339–351.
- 15. Shu CJ, Ulrich LE, Zhulin IB (2003) The NIT domain: a predicted nitrate-responsive module in bacterial sensory receptors. Trends Biochem Sci 28: 121–124.
- 16. Chai W, Stewart V (1999) RNA sequence requirements for NasR-mediated, nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader. J Mol Biol 292: 203–216.
- 17. Bourret RB (2010) Receiver domain structure and function in response regulator proteins. Curr Opin Microbiol 13: 142–149.
- 18. Del Papa MF, Perego M (2008) Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis. J Bacteriol 190: 7147–7156.
- 19. Fox KA, Ramesh A, Stearns JE, Bourgogne A, Reyes-Jara A, et al. (2009) Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis. Proc Natl Acad Sci USA 106: 4435–4440.
- 20. Baker KA, Perego M (2011) Transcription antitermination by a phosphorylated response regulator and cobalamin-dependent termination at a B12 riboswitch contribute to ethanolamine utilization in Enterococcus faecalis. J Bacteriol 193: 2575–2586.
- 21. van Tilbeurgh H, Declerck N (2001) Structural insights into the regulation of bacterial signalling proteins containing PRDs. Curr Opin Struct Biol 11: 685–693.
- 22. Houman F, Diaz-Torres MR, Wright A (1990) Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein. Cell 62: 1153–1163.
- 23. van Tilbeurgh H, Manival X, Aymerich S, Lhoste JM, Dumas C, et al. (1997) Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis. EMBO J 16: 5030–5036.
- 24. Graille M, Zhou CZ, Receveur-Bréchot V, Collinet B, Declerck N, et al. (2005) Activation of the LicT transcriptional antiterminator involves a domain swing/lock mechanism provoking massive structural changes. J Biol Chem 280: 14780–14789.
- 25. Yang Y, Declerck N, Manival X, Aymerich S, Kochoyan M (2002) Solution structure of the LicT-RNA antitermination complex: CAT clamping RAT. EMBO J 21: 1987–1997.
- 26. Morth JP, Feng V, Perry LJ, Svergun DI, Tucker PA (2004) The crystal and solution structure of a putative transcriptional antiterminator from Mycobacterium tuberculosis. Structure 12: 1595–1605.
- 27. Boudes M, Lazar N, Durand D, Gaidenko TA, Stewart V, et al. (2012) Structure of the NasR transcription antiterminator reveals a one-component system with a NIT nitrate-receptor coupled to an ANTAR RNA-binding effector. Mol Microbiol. In press.
- 28. Klocko AD, Wassarman KM (2009) 6S RNA binding to Esigma(70) requires a positively charged surface of sigma(70) region 4.2. Mol Microbiol 73: 152–164.
- 29. Batey RT, Sagar MB, Doudna JA (2001) Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle. J Mol Biol 307: 229–246.