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Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus

AutorKöhler, Karen; Duchardt-Ferner, Elke; Lechner, Marcus; Damm, katrin; Hoch, Philipp G.; Salas, Margarita ; Hartmann, Roland K.
Palabras clave6S RNA
pRNA synthesis
Structure probing
Nuclear magnetic resonance spectroscopy
(NMR)
Hyperthermophilic bacterium
Aquifex aeolicus
Fecha de publicación11-mar-2015
EditorElsevier
CitaciónBiochimie 117: 72- 86 (2015)
Resumen© 2015 Elsevier B.V. and Socie;acuteacute& Franc de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. 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;prime&-CB instead of the 5;prime&-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;prime&-CB.
URIhttp://hdl.handle.net/10261/140495
DOI10.1016/j.biochi.2015.03.004
Identificadoresdoi: 10.1016/j.biochi.2015.03.004
issn: 6183-1638
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