Single-Stranded DNA Aptamers for Functional Probing of Bacterial RNA Polymerase
Bacterial RNA polymerase (RNAP) is the main regulatory hub of gene transcription. During transcription, RNAP interacts with the DNA template, RNA product, nucleotide substrates, metal cofactors, and regulatory molecules that bind to distinct RNAP sites to modulate its activity. RNAP is also inhibited by several known antibiotics and is a promising target for development of novel antibacterial compounds. Despite great progress in structural analysis of RNAP in recent years, many details of RNAP interactions with nucleic acids, regulatory molecules and antibiotics remain insufficiently understood. Aptamers that target various epitopes on the RNAP molecule represent a useful tool for functional analysis of transcription. Here, we describe protocols for selection of highly specific aptamers to different components of RNAP and their applications for analysis of RNAP–ligand interactions and RNAP inhibition.
Key wordsRNA polymerase Aptamers Promoter recognition Transcription inhibition Rifamycin
We thank I. Artsimovitch for plasmids, proteins and rifamycins. This work was supported in part by the Russian Academy of Sciences Presidium Program in Molecular and Cellular Biology and by the Russian Foundation for Basic Research (grants 12-04-33187, 12-04-32042 and 14-04-01696).
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