Since the publication of the classical studies of the Dutch scientist J.D. van der Waals in 1873, physical scientists have known that many—if not most—properties of matter can be rationalized by the strength and direction of the forces that molecules exert on each other. Even strictly macroscopic phenomena, such as the elasticity and the melting points of solids, the viscosity and boiling points of liquids, or the compressibility of gases, are manifestations of the myriad of small interactions between molecules. Chemists have also known that chemical affinity results from the attractive interactions between chemical entities. In 1889, Svante Arrhenius proposed that reactions between molecular species follow pathways that involve the formation of some type of strained, largely unstable, high-energy transition state whose accessibility along the reaction coordinate controls the rate of the reaction.
Until very recently, chemists and biochemists have had to rely on bulk methods to investigate the properties of molecules and their reactions. During the last few years, however, the advent of novel methods of single-molecule manipulation have begun to offer researchers, for the first time, the opportunity to measure directly the forces holding together molecular structures, to measure the stresses and strains generated during the course of chemical and biochemical reactions, to exert external forces to alter the fate of these reactions, and to reveal the rules that govern the interconversion of mechanical and chemical energy in these reactions. This area of research can be rightly called mechanochemistry.
Biochemical processes as diverse as protein folding, DNA elasticity, the protein-induced bending of DNA, the stress-induced catalysis of enzymes, the mechanical properties of molecular motors, and even the ubiquitous processes of induced-fit molecular recognition are all examples in which stresses and strains develop in molecules as they move along a reaction coordinate. My group has played a central role in the development of this field of work.
Sequence-Directed DNA Export Guides Chromosome Translocation During Sporulation in Bacillus subtilis
In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. We used a combination of single-molecule, bioinformatics, and in vivo fluorescence approaches to test these hypotheses. Our data reveal a sequence-directed DNA exporter mechanism that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.
Thermal Probing of Escherichia coli RNA Polymerase Off-Pathway Mechanisms
In an effort to understand the barriers involved in the movement of the enzyme during elongation, we used temperature-controlled optical tweezers to investigate the effect of temperature on the force-velocity behavior of individual RNAP complexes. At temperatures between 7°C and 45°C and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7 ± 0.7 kcal/mol. The pause density at cold temperatures (7ºC–21ºC) was five times higher than that measured above room temperature. Previously, no significant change in the pausing pattern for individual RNAP molecules had been observed within the range studied (21ºC–37ºC). Our results reveal that pause density has a strong temperature dependence below 21ºC and that the previous conclusion reflects only the limited range of temperatures investigated in that study. This dependence has allowed us to obtain important kinetic parameters for the pausing pathway by means of a simple kinetic model. This model revealed a value of 1.29 ± 0.05 kcal/mol for the activation energy of pause entry, indicating that pause entry is indeed a thermally accessible process. The dwell-time distribution of all observable pauses was independent of temperature, confirming a prediction of the model recently proposed for polymerase II, in which pauses result from diffusive backtracking along the DNA. Additionally, the use of a varying force regime revealed that the stall force of the enzyme presents a maximum at 21ºC, an unexpected result, as this is not the optimum temperature for bacterial growth. This suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.
Following Translation by Single Ribosomes One Codon at a Time
Attempts to follow translation by individual ribosomes and characterize their dynamics in real time have been a long-standing quest of biophysicists. To carry out this program, we recently developed a single-ribosome/single-mRNA assay. With this, we have followed individual ribosomes as they translate single mRNA hairpins tethered by the ends to optical tweezers. These studies reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 seconds, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 seconds. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. We also showed that translocation and RNA unwinding (helicase activity) are strictly coupled ribosome functions.
Differential Detection of Dual Traps Improves the Spatial Resolution of Optical Tweezers
The drive toward more sensitive single-molecule manipulation techniques has led to the recent development of optical tweezers capable of resolving the motions of biological systems at the subnanometer level, approaching the fundamental limit set by Brownian fluctuations. One successful approach has been the dual-trap optical tweezers, in which the system of study is held at both ends by microspheres in two separate optical traps. Recently we developed a theoretical description of the Brownian limit on the spatial resolution of such systems and verified these predictions by direct measurement in Brownian noise-limited, dual-trap optical tweezers. By detecting the positions of both trapped microspheres, we could exploit correlations in their motions to maximize the resolving power of the instrument. Remarkably, we demonstrated that the spatial resolution of dual optical traps with dual-trap detection is always superior to that of more traditional, single-trap designs, despite the added Brownian noise of the second trapped microsphere. Our newly built instrument has a signal-to-noise (S/N) = 1 at about 1-Å resolution with a bandwidth of ~50 Hz. This remarkable resolution opens a number of exciting opportunities to follow biochemical reactions at the single molecule level. This ultrahigh resolution instrument is now fully operational, and it is being used in the analysis of several nucleic acid translocases.
