Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

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The cytochrome P-450cam hydroxylase operon of Pseudomonas putida PpG1 (ATCC 17543) encodes proteins responsible for early steps of the degradation of D-camphor. Transcription of this operon is negatively controlled by the cam repressor (CamR), and the expression of camR is autoregulated. CamR was purified from Escherichia coli harboring an overproducing plasmid. The repressor forms a homodimer with a molecular mass of 40 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and gel filtration. CamR protected a specific DNA region from attack by DNase I. This region contains a palindromic operator of the cytochrome P-450cam hydroxylase operon and of the camR gene. Protection was inhibited by the addition of 60 M D-camphor and also by certain camphor analogs and degradation products, including D-3-bromocamphor, adamantane, 2-adamantanone, 5-exo-hydroxycamphor, and 2,5-diketocamphane. These analogs and degradation products induced cytochrome P-450cam hydroxylase operon expression in vivo.The cytochrome P-450cam hydroxylase operon (camDCAB) on the CAM plasmid of Pseudomonas putida PpG1 (ATCC 17453) is responsible for early steps of the D-camphor degradation pathway for catabolism of camphor to isobutyrate (17,18). D-Camphor degradation initiates oxidation to 5-exo-hydroxycamphor by a monooxygenase system which consists of three enzymes: NADH-putidaredoxin reductase (45 kDa; encoded by the camA gene), putidaredoxin (12 kDa; encoded by the camB gene), and cytochrome P-450cam (47 kDa; encoded by the camC gene) (7, 10). The second step is the conversion of alcohol to 2,5-diketocamphane, which is dehydrogenated by 5-exo-hydroxycamphor dehydrogenase (FdeH, 80 kDa; encoded by the camD gene) (14). Expression of the camDCAB operon and the camR gene is negatively regulated through interaction of the CamR protein with the single operator located in the overlapping promoter region between the camD-CAB operon and the camR gene. In the presence of D-camphor, these genes are divergently transcribed from the overlapping promoters (3, 12). When examining structural and functional properties of the CamR repressor, we constructed a heterologous expression system for overproduction of the CamR protein, with Escherichia coli harboring a plasmid for expression of CamR (3).We report here the purification and characterization of the CamR protein from an E. coli culture, in the presence of D-camphor. Both in vitro and in vivo, various camphor analogs inhibited binding of the CamR protein to the operator DNA. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study were E. coli JM83 [FЈ ara ⌬(lac-proAB) rpsL(80lacZ⌬M15)] (21) and P. putida PpG277 (16). A CamR-overproducing plasmid, pHAOV1, was constructed as follows. The translational initiation codon of the camR gene was changed from GTG to ATG to increase translational efficiency, and then the camR gene was placed under the control of a bacteriophage PL promoter of the expression vector pUC-PL-cI (3, 19). Plasmid pHAOV1 wa...

The Eco RI enzyme is a nanomachine responsible for gene integrity in cells, and has a sensor that discriminates its specific binding sequence on DNA (GAATTC) from flanking nonspecific sequences. We applied this sequence sensing ability to microscopic DNA mapping. Visible measurement of positions of the GAATTC sequence was accomplished with fluorescently labeled Eco RI and DNA that was stretched by dielectrophoresis and suspended above a glass surface. Statistics showed that Eco RI molecules were trapped at particular positions on stretched DNA where GAATTC sequences are located. Dwell time at trapped positions agreed with the lifetime of the specific DNA-Eco RI complex previously measured. In a flowing stream, Eco RI molecules moved along stretched DNA and were trapped at putative GAATTC sequences, providing evidence of sliding as a mechanism for relocation of Eco RI on DNA. This single-molecule-based method can be a lab on a chip for mapping genomic DNA and analyzing motility of DNA-binding nanomachines.

The extension of chromosomal DNA is a key technique in high-resolution gene location such as extended fibre fluorescent in situ hybridization. In this paper, we propose a new micro-device designed for on-chip integration of three functions, (1) positioning of cells, (2) extending chromosomal DNA from the cells, and (3) anchoring the extended DNA fibres. The device has a flow chamber equipped with (a) multi-phase electrodes to create electroosmotic flow (EOF) of variable direction, (b) a set of micro-fabricated pocket-like structures for cell positioning, and (c) a micro-pillar array whose surface is chemically modified to anchor DNA fibres. First, unidirectional EOF is induced, by which cells are carried into the micro-pockets and positioned, one cell at each pocket. After rupturing the cells, chromosomal DNA from each cell is hydrodynamically extended by EOF. When the EOF direction is rotated through 90°, the fibres are made to contact with the pillars to be anchored. Because the velocity of the EOF is controllable, the breakage of DNA during the process can be minimized. Thus immobilized DNA fibres are suspended a few microns above the surface, allowing free access of probe molecules.

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A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector-binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. D-camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of D-camphor but, at a higher concentration, is dissociated by the second binding. D-camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of K d1 = 0.064 ± 0.030 and K d2 = 14 ± 0.3 lM, as well as the Hill coefficient of 0.56 ± 0.05 (<1). The first binding guarantees the high specificity of the inducer by the high affinity, although the second binding turns on the gene expression at a 200-fold higher concentration, a more suitable switching point for the catabolism of D-camphor.