title: Next generation sequencing-based parallel analysis of melting kinetics of 4096 variants of a bacterial promoter.
abstract: 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.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School , 1100 S. Grand Boulevard, St. Louis, Missouri 63104, United States.
authors.forename: Ewa
authors.initials: E
authors.lastname: Heyduk
authors.forename: Tomasz
authors.initials: T
authors.lastname: Heyduk
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D014644:Genetic Variation / Q000235:genetics*; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
chemical_list: D009841:Oligonucleotides; D012321:DNA-Directed RNA Polymerases