title: Sequence-Dependent Promoter Escape Efficiency Is Strongly Influenced by Bias for the Pretranslocated State during Initial Transcription.
abstract: Abortive transcription initiation can be rate-limiting for promoter escape and therefore represents a barrier to productive gene expression. The mechanism for abortive initiation is unknown, but the amount of abortive transcript is known to vary with the composition of the initial transcribed sequence (ITS). Here, we used a thermodynamic model of translocation combined with experimental validation to investigate the relationship between ITS and promoter escape on a set of phage T5 N25 promoters. We found a strong, negative correlation between RNAP's propensity to occupy the pretranslocated state during initial transcription and the efficiency of promoter escape (r = -0.67; p < 10(-6)). This correlation was almost entirely caused by free energy changes due to variation in the RNA 3' dinucleotide sequence at each step, implying that this sequence element controls the disposition of initial transcribing complexes. We tested our model experimentally by constructing a set of novel N25-ITS promoter variants; quantitative transcription analysis again showed a strong correlation (r = -0.81; p < 10(-6)). Our results support a model in which sequence-directed bias for the pretranslocated state during scrunching results in increased backtracking, which limits the efficiency of promoter escape. This provides an answer to the long-standing issue of how sequence composition of the ITS affects promoter escape efficiency.
authors.affiliation: †Department of Chemical Engineering, Norwegian University of Science and Technology, Sem Sælandsvei 4, 7491 Trondheim, Norway.
authors.forename: Jørgen
authors.initials: J
authors.lastname: Skancke
authors.affiliation: †Department of Chemical Engineering, Norwegian University of Science and Technology, Sem Sælandsvei 4, 7491 Trondheim, Norway.
authors.forename: Nadav
authors.initials: N
authors.lastname: Bar
authors.affiliation: ‡Department of Biology, Norwegian University of Science and Technology, Høgskoleringen 5, 7491 Trondheim, Norway.
authors.forename: Martin
authors.initials: M
authors.lastname: Kuiper
authors.affiliation: §Program in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts 01075, United States.
authors.forename: Lilian M
authors.initials: LM
authors.lastname: Hsu
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D011401:Promoter Regions, Genetic; D013816:Thermodynamics; D061785:Transcription Initiation, Genetic
chemical_list: D029968:Escherichia coli Proteins; D012321:DNA-Directed RNA Polymerases