A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

Abstract

RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of approximately 3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast

“…Next, we asked whether the protein interaction between Loc1p and She2p is altered by binding to zip-code RNA. For better purification of large amounts of zip-code RNA, we used the tRNA-scaffold technique (29). We fused the 118-bases-long ASH1 E3 element to a tRNA (ASH1 E3-118 tRNA), expressed it in bacteria, and purified it.…”

“…The RNA was phenol-chloroform extracted, ethanol precipitated, and further purified by ion exchange chromatography (DAEA and MonoQ columns), as described in ref. 29. Pure fractions were identified by 8% (vol/vol) Tris/borate/EDTA (TBE) Urea PAGE, pooled, and ethanol precipitated.…”

RNA-methyltransferase TrmA is a dual-specific enzyme responsible forC5-methylation of uridine in both tmRNA and tRNA

“…The second methylated uridine observed in TLD-haa, as explained in text, is most probably U 97 . et al and in Ponchon et al 8,25 Pure fractions of monomeric ectmRNA were separated from aggregates on a Superdex-200 prep grade gel filtration column (GE-Healthcare) equilibrated with 50 mM NaCl, 20 mM potassium phosphate pH 6.5 and 1 mM EDTA water solution. The purity of all samples was checked by 12% PAGE containing 7 M urea.…”

“…The genes of TLDaa, TLD-Haa and ectmRNA were cloned in the pBST-NAV vector (AmpR) between EcoRI and PstI according to the procedure described by Ponchon et al 24,25 The expression of the recombinant RNAs was monitored by carrying out extractions of total soluble RNAs from small-scale overnight cultures in 2XTY growth medium. Large-scale expression was performed overnight at 37°C in 2 L of 2XTY medium containing 100 μg/ml ampicillin.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…A third method used to produce RNA for NMR studies was recently developed by Dardel and coworkers by producing RNA in vivo using a tRNA scaffold to protect the RNA from cellular RNases [121,122]. The tRNA scaffold can be removed either by DNAzymes or by sequence-specific RNase H cleavage [115,[117][118][119].…”