title: Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus.
abstract: Bacterial 6S RNAs competitively inhibit binding of RNA polymerase (RNAP) holoenzymes to DNA promoters, thereby globally regulating transcription. RNAP uses 6S RNA itself as a template to synthesize short transcripts, termed pRNAs (product RNAs). Longer pRNAs (approx. ≥ 10 nt) rearrange the 6S RNA structure and thereby disrupt the 6S RNA:RNAP complex, which enables the enzyme to resume transcription at DNA promoters. We studied 6S RNA of the hyperthermophilic bacterium Aquifex aeolicus, representing the thermodynamically most stable 6S RNA known so far. Applying structure probing and NMR, we show that the RNA adopts the canonical rod-shaped 6S RNA architecture with little structure formation in the central bulge (CB) even at moderate temperatures (≤37 °C). 6S RNA:pRNA complex formation triggers an internal structure rearrangement of 6S RNA, i.e. formation of a so-called central bulge collapse (CBC) helix. The persistence of several characteristic NMR imino proton resonances upon pRNA annealing demonstrates that defined helical segments on both sides of the CB are retained in the pRNA-bound state, thus representing a basic framework of the RNA's architecture. RNA-seq analyses revealed pRNA synthesis from 6S RNA in A. aeolicus, identifying 9 to ∼17-mers as the major length species. A. aeolicus 6S RNA can also serve as a template for in vitro pRNA synthesis by RNAP from the mesophile Bacillus subtilis. Binding of a synthetic pRNA to A. aeolicus 6S RNA blocks formation of 6S RNA:RNAP complexes. Our findings indicate that A. aeolicus 6S RNA function in its hyperthermophilic host is mechanistically identical to that of other bacterial 6S RNAs. The use of artificial pRNA variants, designed to disrupt helix P2 from the 3'-CB instead of the 5'-CB but preventing formation of the CBC helix, indicated that the mechanism of pRNA-induced RNAP release has been evolutionarily optimized for transcriptional pRNA initiation in the 5'-CB.
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: karenkoehler2502@gmail.com.
authors.forename: Karen
authors.initials: K
authors.lastname: Köhler
authors.affiliation: Goethe-Universität Frankfurt am Main, Institut für Molekulare Biowissenschaften, Max-von-Laue-Straße 9, D-60438 Frankfurt am Main, Germany; Zentrum für biomagnetische Resonanzspektroskopie (BMRZ), Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany. Electronic address: duchardt@bio.uni-frankfurt.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Duchardt-Ferner
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: marcus.lechner@staff.uni-marburg.de.
authors.forename: Marcus
authors.initials: M
authors.lastname: Lechner
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: katrin.damm@staff.uni-marburg.de.
authors.forename: Katrin
authors.initials: K
authors.lastname: Damm
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: hoch@Staff.Uni-Marburg.DE.
authors.forename: Philipp G
authors.initials: PG
authors.lastname: Hoch
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Electronic address: msalas@cbm.csic.es.
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
authors.affiliation: Philipps-Universität Marburg, Fachbereich Pharmazie, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg, Germany. Electronic address: roland.hartmann@staff.uni-marburg.de.
authors.forename: Roland K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: C411065:6S RNA; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D012321:DNA-Directed RNA Polymerases
keywords: 6S RNA; Aquifex aeolicus; Hyperthermophilic bacterium; Nuclear magnetic resonance spectroscopy (NMR); Structure probing; pRNA synthesis