In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution

• Published in: RNA (Cambridge) Vol. 20; no. 10; pp. 1607 - 1620
• Main Authors: Ranaei-Siadat, Ehsan, Mérigoux, Cécile, Seijo, Bili, Ponchon, Luc, Saliou, Jean-Michel, Bernauer, Julie, Sanglier-Cianférani, Sarah, Dardel, Fréderic, Vachette, Patrice, Nonin-Lecomte, Sylvie
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01-10-2014
• Subjects: Biochemistry, Molecular Biology; Electrophoretic Mobility Shift Assay; Escherichia coli - metabolism; Life Sciences; Magnetic Resonance Spectroscopy; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Biosynthesis; Protein Conformation; Ribosomes - metabolism; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA-Binding Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structural Biology; X-Ray Diffraction; trans-translation; tmRNA; NMR; SmpB; in vivo protection; SAXS
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.045674.114

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.