Facilitated Target Location on DNA by IndividualEscherichia coli RNA Polymerase Molecules Observed with the Scanning Force Microscope Operating in Liquid

In Escherichia coli, transcription is carried out by a single enzyme, E. coli RNA polymerase (RNAP), and the rate of transcription initiation is controlled, in part, by the rate at which the polymerase can find the promoter. One problem that RNAP, and for that matter most other specific DNA-binding proteins, must solve is how to overcome the kinetic barrier of finding its specific binding site amid a large excess of nonspecific DNA. In 1968, Adam and Delbruck (1) proposed that the efficiency of a diffusion-controlled search could be enhanced by orders of magnitude if it were to take place in a space of lower dimensionality. In 1970, Riggs et al. (2) reported that E. coli lac repressor locates its target site at rates up to 1000 times faster than what could be accounted for by a three-dimensional diffusion-controlled search. These authors suggested that, because the enzyme has some affinity for non-promoter DNA, their observations could be explained if the enzyme could bind nonspecifically anywhere to the DNA and then move in a onedimensional random walk along the DNA until it finds its target. This finding sparked the interest of scientists to demonstrate the existence and to elucidate the mechanisms of facilitated target location in DNA-binding proteins. Two investigations have used kinetic analysis to obtain evidence that RNAP can locate a promoter by one-dimensional diffusion along nonspecific DNA. Singer and Wu (3) employed a rapid mixing/photocross-linking method to monitor the timedependent density of bound RNAP along a relaxed, circular DNA plasmid containing a single promoter. It was found that the occupancy by RNAP of the DNA segments near a promoter decreased faster than the occupancy of the segments farther away from the promoter. This phenomenon was interpreted as evidence that RNAP reached the promoter through one-dimensional diffusion along the DNA. Fitting the data to a theoretical model that included RNAP sliding, a dissociation rate constant of koff 5 0.3 s 21 and a one-dimensional diffusion coefficient of D1D 5 1.5 3 10 29 cm/s for RNAP sliding along DNA were determined. The resulting average lifetime of a nonspecific complex, tav 5 3.3 s, is, however, about 1000 times larger than the one found in electron microscopy experiments (4). In the second study, Ricchetti et al. (5) measured the occupancy of DNA fragments carrying A1 promoters as a function of the length of the downstream and upstream flanking sequences. It was found that a longer downstream flanking sequence increased the occupancy of the adjacent promoter in agreement with the sliding model. However, upstream sequences had surprisingly little effect on promoter occupancy. In two recent studies, fluorescence microscopy was used to observe the interactions of fluorescently labeled RNAP with DNA. Kabata et al. (6) utilized superintensified fluorescence microscopy to visualize the movement of RNAP over l-DNA combs. A fraction of the RNAP molecules was seen to deviate from the direction of bulk flow and to move along the extended DNA molecules. This observation suggests that RNAP can slide along nonspecific DNA. However, in this experiment the RNAP was propelled predominantly by flow and was, therefore, not driven by thermal motion. In the second study, Harada et al. (7) used internal reflection fluorescence microscopy to observe the dissociation and association events of RNAP with different regions of a single l-DNA molecule, which was suspended in laser tweezers. For AT-rich regions fast and slow dissociation constants of 3.0 and 0.66 s, respectively, were determined; and for GC-rich regions a fast dissociation rate of 8.4 s was measured. In a few instances sliding of RNAP along the DNA was also observed. However, the occurrence of these events was rare because the present spatial resolution of this technique is about 200 nm, and the typical sliding range of a one-dimensionally diffusing RNAP is presumably less than this limit. Nevertheless, a one-dimensional diffusion constant of ;10 cm/s was estimated from these data. Other mechanisms of facilitated target location besides sliding have been proposed, as depicted in Fig. 1. One mechanism involves the transfer of DNA-binding proteins from one segment of DNA to another. This process, known as intersegment transfer (8), could make it more likely for a protein molecule to bind to distant regions in the genome by decreasing the effective volume of diffusion and thus increasing the target location rate. Moreover, as pointed out by von Hippel and Berg (8), the efficiency of this process can be enhanced if the energy barrier of intersegment transfer is lower than the barrier for the dissociation-association cycle. In particular, the high local concentration of DNA would also make contact increasingly likely and multiple transfer events possible. A third proposed mechanism is the intradomain association and dissociation or “hopping,” of the protein along the DNA (8). In this process, the protein-DNA interactions may consist of the protein bouncing along the DNA until it finds the target site or completely dissociates from the DNA. The target search would then be accelerated in a manner similar to sliding, with a reduced number of sampled sequences but presumably higher kinetic barriers.