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

Abstract

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.

Structures of tmRNA and SmpB as they transit through the ribosome

“…The tmRNAs were purified in native conditions as previously described 60 . We used a synthetic non-stop mRNA (Thermo Fisher Scientific) with the sequence 5′-AGGAGGUGAGGU UUU -3′ (the Shine–Dalgarno sequence is underlined and the phenylalanine codon is bold).…”

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs

“…Therefore, there is a need to develop novel technologies, especially microbial fermentation-based methods, for the production of biologic RNA molecules (Ho and Yu 2016;Pereira et al 2017;Yu et al 2019) that allow for cellular machineries to recognize and perform post-transcriptional modification and processing to necessary structures and folding. Indeed, recombinant RNAs or BERAs have none or just minimal post-transcriptional modifications, such as pseudouridine (Gaudin et al 2003;Li et al 2015;Nelissen et al 2012;Ponchon et al 2009;Ponchon and Dardel 2007;Ranaei-Siadat et al 2014;Wang et al 2015), which are necessary to resemble natural RNAs and pose intrinsic secondary and high-order structures. Furthermore, bioengineered RNA molecules produced heterogeneously in microbial fermentation have been demonstrated to be biologically functional in vitro and in vivo by various studies (Chen et al 2015;Ho et al 2018;Jian et al 2017;Jilek et al 2019;Li et al 2014Li et al , 2015Li et al , 2018Li et al , 2019Liu et al 2010;Nelissen et al 2012;Paige et al 2011Paige et al , 2012Pereira et al 2016a, b;Pitulle et al 1995;Tu et al 2019;Wang et al 2015;Zhang et al 2009;Zhao et al 2016).…”