Cotranscriptional Assembly and Native Purification of Large RNA-RNA Complexes for Structural Analyses

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.

Keywords: Cotranscriptional folding; RNA–RNA interactions; Riboswitches; T-box; Transcription; tRNA.

Fig. 1.. Size-exclusion chromatography (SEC) purification of a T-box discriminator – tRNA^Gly complex.

(a) Secondary structure of Geobacillus kaustophilus glyQ T-box Discriminator – tRNA^Gly complex informed by the co-crystal structure (PDB: 6PMO). Inset: cartoon scheme of a typical full-length glycine-specific T-box riboswitch bound to its cognate tRNA substrate. Dotted lines indicate stacking interactions between the apical region of the Stem I and tRNA elbow. (b) SEC profile of native, co-transcriptionally folded and assembled T-box Discriminator – tRNA^Gly complex. Main peaks are labeled, and their contents indicated. (c) Denaturing gel (10%) analysis of peak fractions of (b). Bands are annotated. (d) Non-denaturing gel (10%) analysis of the same peak fractions in (b) & (c).

Fig. 2.. Anion-exchange chromatography purification of a full-length T-box – tRNA^Gly complex.

(a) Secondary structure of Bacillus subtilis glyQS full-length T-box– tRNA^Gly complex informed by two co-crystal structures (PDB: 6PMO; 4LCK) and cryo-EM structure (PDB: 6POM). (b) Mono Q profile of native, co-transcriptionally folded and assembled full-length T-box– tRNA^Gly complex. Main peaks are labeled, and their contents indicated. The conductivity trace representing the salt gradient is shown in orange. (c) Denaturing gel (10%) analysis of peak fractions of (b). Bands are annotated. (d) Non-denaturing gel (10%) analysis of the same peak fractions in (b) & (c).

Fig. 3.. Crystal and Cryo-EM structures solved using the protocol and corresponding method details.

(a) Co-crystal structure of the Geobacillus kaustophilus glyQ T-box Discriminator – tRNA^Gly complex (PDB: 6PMO). (b) Co-crystal structure of the Nocardia farcinica ileS T-box Stem I-Stem II domains in complex with the cognate tRNA^Ile (PDB: 6UFM). (c) Cryo-EM structure of the Bacillus subtilis glyQS T-box full-length RNA (anti-terminated conformation) in complex with its cognate tRNA^Gly (PDB:6POM). (d) Characteristics of RNA samples and method parameters employed to solve the structures in (a-c).