Martin Guthold

Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Controlled manipulation of molecular samples with the nanoManipulator

The nanoManipulator system adds a virtual-reality interface to an atomic-force microscope (AFM), thus providing a tool that can be used by scientists to image and manipulate nanometer-sized molecular structures in a controlled manner. As the AFM tip scans the sample, the tip-sample interaction forces are monitored, which, in turn, can yield information about the frictional, mechanical, material, and topological properties of the sample. Computer graphics are used to reconstruct the surface for the user, with color or contours overlaid to indicate additional data sets. Moreover, a force feedback stylus, which is connected to the tip via software, allows the user to directly interact with the macromolecules. This system is being used to investigate carbon nanotubes, DNA, fibrin, adeno- and tobacco mosaic virus. It is now also possible to insert this system into a scanning electron microscope which provides the user with continuous images of the sample, even while the AFM tip is being used for manipulations.

Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope

A simple method of substrate preparation for imaging circular DNA molecules with the scanning force microscope (SFM) is presented. These biomolecules are adsorbed onto mica that has been soaked in magnesium acetate, sonicated and glow-discharged. The stylus-sample forces that may be endured before sample damage occurs depends on the ambient relative humidity. Images of circular DNA molecules have been obtained routinely using tips specially modified by an electron beam with a radius of curvature, Rc, of about 10 nm [D. Keller and C. Chih-Chung, Surf. Sci. 268 (1992) 333]. The resolution of these adsorbed biomolecules is determined by the Rc. At higher forces individual circular DNA molecules can be manipulated with the SFM stylus. Strategies to develop still sharper probes will be discussed.

DNA-functionalized single-walled carbon nanotubes

We present here the use of amino-terminated DNA strands in functionalizing the open ends and defect sites of oxidatively prepared single-walled carbon nanotubes, an important first step in realizing a DNA-guided self-assembly process for carbon nanotubes.

Increased heating efficiency and selective thermal ablation of malignant tissue with DNA-encased multiwalled carbon nanotubes.

Nanoparticles, including multiwalled carbon nanotubes (MWNTs), strongly absorb near-infrared (nIR) radiation and efficiently convert absorbed energy to released heat which can be used for localized hyperthermia applications. We demonstrate for the first time that DNA-encasement increases heat emission following nIR irradiation of MWNTs, and DNA-encased MWNTs can be used to safely eradicate a tumor mass in vivo. Upon irradiation of DNA-encased MWNTs, heat is generated with a linear dependence on irradiation time and laser power. DNA-encasement resulted in a 3-fold reduction in the concentration of MWNTs required to impart a 10 degrees C temperature increase in bulk solution temperature. A single treatment consisting of intratumoral injection of MWNTs (100 microL of a 500 microg/mL solution) followed by laser irradiation at 1064 nm, 2.5 W/cm(2) completely eradicated PC3 xenograft tumors in 8/8 (100%) of nude mice. Tumors that received only MWNT injection or laser irradiation showed growth rates indistinguishable from nontreated control tumors. Nonmalignant tissues displayed no long-term damage from treatment. The results demonstrate that DNA-encased MWNTs are more efficient at converting nIR irradiation into heat compared to nonencased MWNTs and that DNA-encased MWNTs can be used safely and effectively for the selective thermal ablation of malignant tissue in vivo.

Wrapping of DNA around the E.coli RNA polymerase open promoter complex

High‐resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase·σ70 (RNAP) open promoter complex (RPo). A detailed analysis of a large number of molecules shows that the DNA contour length of RPo is reduced by ∼30 nm (∼90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88°. The contour length reduction and the DNA bend angle were much less in inactive RNAP–DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300° around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two‐thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one‐third is part of the downstream region. Based on these data, a model of the σ70·RPo conformation is proposed.

A Comparison of the Mechanical and Structural Properties of Fibrin Fibers with Other Protein Fibers

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young’s modulus, E) and breaking strain (also called rupture strain or extensibility, εmax) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young’s modulus on the order of a few Gigapascal and are not very extensible (εmax < 20%). They also display a very regular arrangement of their monomeric units. Soft biological protein fibers have a Young’s modulus on the order of a few Megapascal and are very extensible (εmax > 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an α-helix to β-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the γ-chain.

The mechanical properties of single fibrin fibers

See also Weisel JW. Biomechanics in hemostasis and thrombosis. This issue, pp 1027–9. Carlisle CR, Sparks EA, Der Loughian C, Guthold M. Strength and failure of fibrin fiber branchpoints. This issue, pp 1135–8.

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.

The mechanical properties of individual, electrospun fibrinogen fibers

We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 microm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 x 10 (6)Pa. We collected incremental stress-strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 x 10 (6)Pa and the relaxed elastic modulus was 7.2 x 10 (6)Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s. Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that alpha-helical content of fibrinogen is approximately 70% higher in 90-HFP than in aqueous solution. These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.