One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location.

Abstract

The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated. The occupancies of DNA fragments carrying the Al promoter of bacteriophage 17 were analyzed as a function of the length of flanking sequences adjacent to the promoter. Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy. This was studied quantitatively by using a set of DNA fragments with four identical Al promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV. The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy. Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion.Location of a DNA signal sequence by facilitated diffusion (1-4) has been proposed for a number of sequence-specific binding proteins. For example, there is good evidence in the cases of lac repressor (5, 6), EcoRI restriction endonuclease (7,8), and DNA polymerase (9) for mechanisms that compress the three-dimensional diffusion volume. Such mechanisms involve formation of a nonspecific protein-DNA complex and subsequent diffusion of the protein within or along the DNA domain until either the signal sequence is located or dissociation occurs. Translocation within the DNA domain results in a positionally uncorrelated search pattern, where the protein either dissociates from the DNA microscopically and probes adjacent sites on the DNA ("hopping") (4), or dissociates macroscopically and rebinds to a new site. This paper addresses two questions: (i) to what extent do one-dimensional diffusion processes participate in the location of a promoter and (ii) is this process directional? Sliding as a means of accelerating the promoter-search process by DNA-dependent RNA polymerase (EC 2.7.7.6) was proposed in 1982 by von Hippel et al. (10). We have designed a system that allowed us to test this idea by means of competition experiments in which the occupancies of promoters with flanking DNA sequences of different lengths were compared. Our hypothesis was that nonspecific DNA sequences serve as "antennae" (10) along which the Escherichia coli RNA polymerase moves to the promoter. If this antenna effect exists, the occupancy of a promoter should be influenced by the length of the flanking sequences. tests (17). The concentration of the DNA was determined by UV absorption (assuming an extinction coefficient at 260 nm of 20 cm2/mg). EXPERIMENTAL PROCEDURESMethods. Binding of RNA polymerase (3 ,AM) to promotercarrying DNA fragments (3 ,...

Spatial Distribution and Diffusive Motion of RNA Polymerase in Live Escherichia coli

“…On average, roughly half the population of RNAP molecules is mobile over the 2-to 3-m length of the cell on a timescale of 5 to 10 s, with an effective diffusion constant D RNAP of approximately 0.2 m 2 -s Ϫ1 . Remarkably, the D RNAP is comparable to diffusion constants measured in vitro for RNAP sliding along the contour of bare, double-stranded DNA (dsDNA) while nonspecifically bound (10,26,30). The other half of the population is immobile on a timescale of at least 30 to 60 s. We attribute the immobile fraction to RNAP copies that are specifically bound to DNA; at least some of these copies are presumably actively transcribing DNA.…”

RNA polymerase approaches its promoter without long-range sliding along DNA

“…Thus, adding the long extension does not detectably increase promoter binding; the data imply that no more than 8% of the long-lived promoter binding events on the long DNA could arise from sliding that originated in the long downstream segment (SI Materials and Methods). A previous study suggested that truncation of segments upstream or downstream had differing effects on promoter occupancy by σ 70 RNAP (20). In our earlier work (7), we truncated the DNA upstream of the glnAp2 promoter (instead of downstream) and measured the effects of the truncation on the kinetics of σ 54 RNAP binding and release using the same methods as in Fig.…”

“…Therefore, σ 54 RNAP reaches its target sequence by a mechanism that differs from the mechanism used by the transcription factors and DNA binding proteins that have been observed to reach their targets by sliding (41)(42)(43). Many previous studies on E. coli RNAP have been interpreted as supporting an FD model for promoter search with sliding distances of hundreds to thousands of base pairs (13)(14)(15)(16)(17)(18)(19)(20)(21). Those studies used a different holoenzyme (σ 70 instead of σ 54 ), and many were conducted at unphysiologically low ionic strength; therefore, it is possible that promoter search by FD occurs under the conditions of those experiments but not our experiments.…”

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

“…RNA polymerase binds to DNA and diffuses one-dimensionally to the promoter region (1)(2)(3)(4); it then must make a number of interactions within the promoter as a prerequisite to forming a relatively stable complex. This complex is then in a state that favors isomerization, leading to strand separation from approximately Ϫ12 to ϩ3 with respect to the site of transcription initiation (5).…”