Sequencing-based mutational analysis of RNA transcription termination (SMARTT). (A) Schematic of the strategy used for the high-throughput mutational analysis of transcription termination by sequencing. Mutant libraries were made by error-prone PCR (hypothetical mutations shown in yellow) and used as DNA template for a series of in vitro transcription reactions containing varying concentrations of glycine. RNA obtained from these reactions was prepared and sent for high-throughput sequencing (sequencing adapters are shown in cyan). The resulting sequences were computationally analyzed to produce response profiles for all variants with 0–2 mutations simultaneously. Representative response profiles are shown for mutants that alter the parameters Ymin (B), Ymax (C), and K1/2 (D). Error bars representing the standard deviation do not appear as they would be smaller than the size of each point.
