PTT-quant - a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches.

Abstract

Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism is hampered by lack of highly sensitive and accurate experimental methods. Here we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of SAM riboswitch induction, which turned to be [~]23% higher in comparison the results obtained without cDNA synthesis normalization.