Abstract
This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The first part of the protocol also describes the in vitro transcription and labeling of the tRNA.
Keywords: T-box, EMSA, RNA-RNA interaction, TRNA, In vitro transcription
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Scanned image of denaturing Urea-PAGE of labelled tRNAs. This example shows dominant single products for most tRNA IVT reactions (arrow), but also a diffuse pattern of several products in one sample (lane 5). This sample would result in an unspecific binding reaction, thus the IVT must be repeated or the correct band eluted from the gel. lane 1: size marker; lane 2 - 6: different in vitro transcribed tRNAs. Figure 2. Scanned image of native PAGE gel after binding assay. This is an example of the separation of unbound, small tRNA* at the bottom of the gel and the tRNA* bound to the much bigger T-box RNA (arrow), which was transcribed in vitro in the presence of pre-formed, radiolabelled(*) tRNA. lane 1: control IVT reaction of T-box RNA* without tRNA; lane 2: T-box RNA IVT with tRNA*fMet; lane 3: T-box RNA IVT with tRNA*Cys (not binding to T-box RNA).
Notes
Recipes
Acknowledgments
This work was funded by the German Research Foundation (DFG), Transregio34 “Pathophysiology of staphylococci in the post-genomic era”, and my PhD stipend was granted by the European Social Fund (ESF). The protocol presented here was developed in the lab of PD Dr. Wilma Ziebuhr during my research time at the Queen’s University Belfast (QUB).
References
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