Abstract
Recent technological developments such as cryogenic electron microscopy (Cryo-EM) and X-ray free electron lasers (XFEL) have significantly expanded the available toolkit to visualize large, complex noncoding RNAs and their complexes. Consequently, the quality of the RNA sample, as measured by its chemical monodispersity and conformational homogeneity, has become the bottleneck that frequently precludes effective structural analyses. Here we describe a general RNA sample preparation protocol that combines cotranscriptional RNA folding and RNA–RNA complex assembly, followed by native purification of stoichiometric complexes. We illustrate and discuss the utility of this versatile method in overcoming RNA misfolding and enabling the structural and mechanistic elucidations of the T-box riboswitch–tRNA complexes.
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References
Harris KA, Breaker RR (2018) Large noncoding RNAs in bacteria. Microbiol Spectr 6:6.4.01.
Peselis A, Gao A, Serganov A (2015) Cooperativity, allostery and synergism in ligand binding to riboswitches. Biochimie 117:100–109
Reiter NJ, Osterman A, Torres-Larios A et al (2010) Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA. Nature 468:784–789
Suslov NB, Dasgupta S, Huang H et al (2015) Crystal structure of the Varkud satellite ribozyme. Nat Chem Biol 11:840–846
Ferré-D’amaré AR, Scott WG (2010) Small self-cleaving ribozymes. In: Gesteland RF, Cech TR, Atkins JF (eds) The RNA world, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Korostelev A, Trakhanov S, Laurberg M et al (2006) Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126:1065–1077
Ramakrishnan V (2002) Ribosome structure and the mechanism of translation. Cell 108:557–572
Bou-Nader C, Zhang J (2020) Structural insights into RNA dimerization: motifs, interfaces and functions. Molecules 25:2881
Hood IV, Gordon JM, Bou-Nader C et al (2019) Crystal structure of an adenovirus virus-associated RNA. Nat Commun 10:2871
Zhang J, Ferre-D’amare AR (2014) Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22:1363–1371
Stagno JR, Liu Y, Bhandari YR et al (2017) Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541:242–246
Somarowthu S, Legiewicz M, Chillon I et al (2015) HOTAIR forms an intricate and modular secondary structure. Mol Cell 58:353–361
Wickiser JK, Winkler WC, Breaker RR et al (2005) The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49–60
Zhang J, Lau MW, Ferré-D’amaré AR (2010) Ribozymes and riboswitches: modulation of RNA function by small molecules. Biochemistry 49:9123–9131
Zhang J, Landick R (2016) A two-way street: regulatory interplay between RNA polymerase and nascent RNA structure. Trends Biochem Sci 41:293–310
Wong TN, Sosnick TR, Pan T (2007) Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures. Proc Natl Acad Sci USA 104:17995–18000
Zhang J (2020) Unboxing the T-box riboswitches-A glimpse into multivalent and multimodal RNA-RNA interactions. Wiley Interdiscip Rev RNA 11:e1600
Li S, Su Z, Lehmann J et al (2019) Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions. Nat Struct Mol Biol 26:1094–1105
Suddala KC, Zhang J (2019) High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122
Coleman TM, Wang G, Huang F (2004) Superior 5′ homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter. Nucleic Acids Res 32:e14
Martin CT, Coleman JE (1989) T7 RNA polymerase does not interact with the 5′-phosphate of the initiating nucleotide. Biochemistry 28:2760–2762
Vasilyev N, Serganov A (2016) Preparation of short 5′-triphosphorylated oligoribonucleotides for crystallographic and biochemical studies. Methods Mol Biol 1320:11–20
Ferré-D’amaré AR, Doudna JA (1996) Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res 24:977–978
Xiao H, Murakami H, Suga H et al (2008) Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454:358–361
Kao C, Zheng M, Rudisser S (1999) A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5:1268–1272
Zhang J, Ferré-D’amaré AR (2014) Direct evaluation of tRNA aminoacylation status by the T-box riboswitch using tRNA-mRNA stacking and steric readout. Mol Cell 55:148–155
Acknowledgments
We thank G. Piszczek and D. Wu for support with biophysical analyses, and A. R. Ferré-D’Amaré, K. Suddala, and C. Bou-Nader for discussions. This work was supported by the Intramural Research Program of the NIH, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (ZIADK075136 to J.Z.), and an NIH Deputy Director for Intramural Research (DDIR) Challenge Award to J.Z. The authors declare no conflicts of interest.
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Sapkota, K.P., Li, S., Zhang, J. (2023). Cotranscriptional Assembly and Native Purification of Large RNA–RNA Complexes for Structural Analyses. In: Ding, J., Stagno, J.R., Wang, YX. (eds) RNA Structure and Dynamics. Methods in Molecular Biology, vol 2568. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2687-0_1
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DOI: https://doi.org/10.1007/978-1-0716-2687-0_1
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