tor hairpin stem [25]. This stem is interrupted by two non-identical asym- metric internal loops, which are recog- nized similarly by the protein dimer. Sequence analysis suggests that this RNA-binding mode likely is conserved in homologous systems [25]. 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 [13]; Mycobacterium tuberculosis Rv1626, receiver [26]; and NasR, NIT [27]. Each protein contains an a-helical coiled-coil connecting the two References 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 39- 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 termina- tion/antitermination decisions. Bio Essays 24: 700–707. 4. Roth A, Breaker RR (2009) The structural and functional diversity of metabolite-binding ribos- witches. Annu Rev Biochem 78: 305–334. 5. Gutie´rrez-Preciado A, Henkin TM, Grundy FJ, Yanofsky C, Merino E (2009) Biochemical features and functional implications of the RNA-based T-box regulatory mechanism. Micro- biol 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 expres- sion. PLoS Genet 8: e1002666. doi:10.1371/ journal.pgen.1002666. 7. Drew R, Lowe N (1989) Positive control of Pseudomonas aeruginosa amidase synthesis is medi- ated by a transcription anti-termination mecha- nism. J Gen Microbiol 135: 817–823. 8. Wilson SA, Wachira SJM, Norman RA, Pearl LH, Drew RE (1996) Transcription anti- termination regulation of the Pseudomonas aerugi- nosa amidase operon. EMBO J 15: 5907–5916. 9. Goldman BS, Lin JT, Stewart V (1994) Identifi- cation and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon ex- pression 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. 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 [6]. The ANTAR domain adopts a helix- turn-helix conformation, but how it binds RNA is unknown. RNA-binding helix- turn-helix proteins include s70 region 4.2, which binds the 235 region of promoter DNA as well as 6S RNA [28], and Ffh, which binds 4.5 S RNA in the signal recognition particle [29]. Both of these RNA targets form bulged stem-loop struc- 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 tran- scription 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-respon- sive 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 posttran- scriptional 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. tures, so the binding sites have character- istics of dsRNA. Future progress in understanding AN- TAR-mediated antitermination requires knowledge of ANTAR domain conforma- tion 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. Acknowledgments We thank Prof. Karen Wassarman for helpful discussion. 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-Bre´chot V, Collinet B, Declerck N, et al. (2005) Activation of the LicT transcriptional antiterminator in- volves a domain swing/lock mechanism provok- ing 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 antitermi- nator 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-com- ponent 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 recog- nition by a universally conserved protein from the signal recognition particle. J Mol Biol 307: 229–246. PLoS Genetics | www.plosgenetics.org 3 June 2012 | Volume 8 | Issue 6 | e1002773