On the promoter complex formation rate of E.coli RNA polymerases with T7 phage DNA

Abstract

Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is discussed.

One-dimensional Diffusion of Proteins along DNA

“…A similar controversy was found in the binding of E. coli RNA polymerase (15). Since then kinetic evidence for sliding has been accumulated by using the assays shown in Fig.…”

“…The filterbinding technique has been widely used (14,15,18,24,26,29,31,34,37) but is open to misinterpretation if there are two or more types of complexes that are trapped with different efficiencies (37). If practicable, the gel shift assay (38,39) is an excellent choice (24,37,40), because it can be made highly quantitative by introducing a competition between two DNA fragments (41).…”

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…In 1970, LacI was reported to bind to the lac operator site at rates 1000-fold faster than could be explained by random, diffusional encounters with the DNA in three dimensions (37), and an analogous phenomenon has also been reported for RNAP (38). Two independent mechanisms, sliding and intersegment transfer, have been proposed to account for the enhanced binding rates.…”

Facilitated diffusion of a DNA binding protein on chromatin.

“…Bacterial DNA binding proteins as diverse as the lac repressor Von Hippel et al, 1982), RNA polymerase Belintsev et al, 1980;Park et al, 1982) and the restriction endonuclease EcoRI (Jack et al, 1982), locate specific DNA sequences in vitro by facilitated diffusion, whereby the search is confined within the molecular domain rather than throughout the bulk solution. We have used Eschenchia coli RNA polymerase as a test protein to discover whether a similar mechanism can operate in chromatin and whether there are permissive and non-permissive states of chromatin structure with respect to such migration.…”

“…No such evidence exists for the eukaryotic polymerase. The initi-ation of RNA chains is confined to sites in the linker regions of chromatin (Williamson and Felsenfeld, 1978;Wasylyk et al, 1979;Hannon et al, 1984), which are short relative both to the separation between promoter sites (Cedar and Felsenfeld, 1973;Hannon et al, 1984) and the mean free diffusional path of the enzyme on DNA (Belintsev et al, 1980) in the conditions of initiation complex formation in vitro. The enzyme must therefore on average explore a considerable number of linker segments before finding a promoter site.…”