Abstract
In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin–triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.
Highlights
The preparation of defined transcription elongation complexes (TECs) is important for in vitro biochemical, biophysical, and structural studies of RNA polymerases (RNAPs) and nascent RNA [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]
I previously observed that positioning E. coli RNAP at an internal desthiobiotin–triethylene glycol (TEG) stall site blocks attachment to streptavidin-coated magnetic beads [27]
If biotin–TEG is not sequestered by a TEC, the DNA is attached to a magnetic bead by both biotin–TEG and 5’ PC biotin
Summary
Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads. Strobel* From the Department of Biological Sciences, The University at Buffalo, Buffalo, New York, USA
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