Next generation sequencing-based parallel analysis of melting kinetics of 4096 variants of a bacterial promoter.

Promoter melting by bacterial RNA polymerase is a key step in transcription initiation. We used a next generation sequencing (NGS) based approach to analyze in parallel promoter melting of all 4096 sequence variants of the 6 bp -10 promoter element. We used NGS read count for each sequence of a promoter library containing a randomized -10 sequence as an observable to determine relative enrichment of -10 element sequence variants at different time points of the promoter melting reaction. The analysis reinforced the dominating role of consensus bases at positions -11 and -7, demonstrated an enhanced preference for A at -11 among sequences exhibiting the fastest melting kinetics, and showed higher overall importance of the T at -7 compared to the A at -11 for efficient promoter melting. Sequences lacking the consensus bases at -7 or -11 could still melt fast if they contained compensatory base patterns at other positions. We observed a significant correlation between the duplex melting energy of -10 element and the kinetics of promoter melting that became more pronounced when the dominating base-specific interactions with RNAP were diminished. These observations indicate that promoter melting kinetics is determined by a combination of base-specific effects/interactions and sequence-dependent stability of DNA duplex with the former playing a dominating role. Our data show that NGS can provide a reliable, quantitative readout for a highly parallel analysis of DNA template sequence dependence of activities of proteins that bind or operate on a DNA template.