Molecular mechanism of promoter selection in gene transcription. II. Kinetic evidence for promoter search by a one-dimensional diffusion of RNA polymerase molecule along the DNA template.

The rapid mixing-photocrosslinking technique, in conjunction with an immunoprecipitation assay developed to measure the change in the distribution of Escherichia coli RNA polymerase molecules bound to T7 DNA, has been applied to investigate the molecular mechanism of promoter search by RNA polymerase. The binding of RNA polymerase to the DNA template can be divided into at least two steps. The initial binding is rapid and occurs at nonspecific sites randomly distributed throughout the DNA molecule. This is followed by a relatively slow promoter search in which RNA polymerase is transferred from nonspecific sites to promoter sites through a series of intramolecular processes. The rate of polymerase loss from a segment of DNA which does not contain promoter sites is a function of the distance from this segment to both the promoter sites and the ends of the DNA molecule. The kinetic data are consistent with a molecular mechanism in which RNA polymerase undergoes a bidirectional linear diffusion along the DNA template to search for the promoter site. This interpretation is supported by the computer simulation which correctly predicts the relative rates of polymerase loss from various DNA segments. The mechanism derived from these studies is in accordance with the notion that the whole DNA molecule serves as an effective sink for trapping and guiding polymerase molecules during promoter search.