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Mechanical Studies of Single Molecules
Summary: Carlos Bustamante uses novel methods of single-molecule visualization, such as scanning force microscopy, to study the structure and function of nucleoprotein assemblies. His laboratory is developing methods of single-molecule manipulation, such as optical and magnetic tweezers, to characterize the elasticity of DNA, to induce the mechanical unfolding of individual protein and RNA molecules, and to investigate the machine-like behavior of molecular motors.
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, the stresses and strains generated during the course of chemical and biochemical reactions, and the effect of external forces on the fate of these reactions, and to reveal the rules that govern the interconversion of mechanical and chemical energy in these processes. We are using these methods to study various nucleic acid–binding molecular motors.
DNA Packaging by Bacteriophage Phi29
The question of how the double-stranded DNA (dsDNA) bacteriophages get their genomic DNA inside their capsid has concerned biologists ever since Salvador Luria and Thomas Anderson made the first electron micrographs of phage T2 and saw tadpole-shaped objects that they likened to spermatozoa. Initially, it seemed obvious that the DNA must first condense into a compact form, after which the capsid proteins would come together to form a shell around it. The problem was framed in its current form about 25 years ago when it was shown—astonishingly at the time—that during the latter stages of a phage infection, an empty protein shell is assembled first and then the DNA is somehow transported across the shell into the interior. Soon it was shown that a tiny motor placed at the base of the capsid carries out the job of packaging the DNA. This mechanism is common to other tailed bacteriophages and animal viruses such as herpes, poxvirus, and adenovirus.
The DNA-packaging motor of bacteriophage Phi29 is made up of three concentric rings (see Figure 1): (1) the head-tail connector, a dodecamer that fits in the pentameric opening at one of the ends of the capsid; (2) a ring of five molecules of RNA, each 174 nt long of unknown function; and (3) a pentameric ring of gp16, an ATPase that belongs to the FtsK/HerA superfamily of ASCE (additional strand, conserved E) ATPases. Because many other processes, such as DNA recombination, prokaryotic chromosome segregation, and gene transposition, involve the movement of DNA or RNA by ATP-driven ring motors belonging to the ASCE/AAA+ superfamily, the study of the Phi29 system should shed light into the molecular mechanisms of other members of this important group of proteins.
Using the experimental design shown in Figure 2A, we previously established that the motor is one of the strongest described to date, capable of producing forces as high as 60 piconewtons (pN), corresponding to an internal pressure of DNA inside the capsid at the end of packaging of ~6 MPa, or 60 atm. Moreover, we later showed that the power stroke of the ATPases coincides with the release of inorganic phosphate from ATP hydrolysis. We also showed that the activities of the ATPases around the ring are coordinated with each other. Thus, addition of small amounts of nonhydrolyzable ATP analogs pauses the motor for variable times, presumably the time required by the ATPase to exchange its nonhydrolyzable analog for ATP. The density of pauses (pauses per unit length of DNA packaged) increases linearly with the concentration of analog, indicating that a single bound analog is sufficient to stop the motor.
Resolving the individual steps of the packaging motor. We recently reported the first direct observation of the intersubunit coordination and the step size of a ring ATPase. Using ultra-high-resolution optical tweezers (Figure 3A), we have established that packaging occurs in increments of 10 bp separated by stochastically varying dwell times (Figure 3B). We also confirmed our previous observation that the ATPases bind ATP with a Hill coefficient of 1. Statistical analysis of the dwell times preceding these steps reveals that multiple ATPs bind during each dwell; application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps (Figure 4A,B). These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated via a mechanism novel for ring ATPases, wherein ATP binding occurs during the "dwell" phase that is completely segregated from the translocation or "burst" phase (Figure 3B). Moreover, a step size that is a noninteger number of base pairs require motor-DNA interactions that do not depend on any given periodic structure in the DNA molecule and that are of steric nature.
The nature of the DNA-motor interactions. Relatively little is known about how these ring motors engage their nucleic acid substrates. In particular, we wish to rationalize the large forces generated by these phages and the 2.5-bp step size of Phi29. To investigate the role of the phosphate backbone charge, we tested the ability of the motor to package DNA constructs containing inserted regions of uncharged methylphosphonate (MeP) modifications. Remarkably, the motor actively traverses these inserts, though with reduced probability compared to native DNA, indicating that phosphate charges are important but not essential for translocation. By changing the length of the MeP inserts and restoring the charge to one or the other DNA strand selectively, we determined that important contacts are made with phosphates on the 5'→3' strand every 10 base pairs. High-resolution packaging trajectories through the insert reveal that, in addition to providing a load-bearing contact, these phosphates play a role in regulating the mechanochemical cycle.
Transcription through Nucleosomal DNA
A second area of active research is the study of transcription elongation by eukaryotic RNA polymerase (RNAP). We have investigated the physical bases of transcription regulation by nucleosomes. We followed individual RNAPII elongation complexes as they transcribe a DNA template wrapped in a nucleosome. We found that a nucleosome acts as a mechanical barrier that increases the local pause density and arrest probability of the polymerase in a strong ionic strength-dependent manner (Figure 5). The distribution of nucleosome-induced pauses reveals that recovery from transcriptional pausing is slowed in the presence of a nucleosome. We quantitatively model the effect of the nucleosomal barrier on the pause density, arrest, and backtrack recovery probability by assuming that the polymerase is unable to actively separate the DNA from its wrapping around the core histone and must, therefore, await transient local openings of the nucleosome. In addition, we follow the fate of single nucleosomes immediately after the passing of RNAPII. We found that the process of histone transfer is extremely sensitive to tension in the DNA upstream of the polymerase, supporting a model of histone transfer that requires the formation of a (force-sensitive) transient loop with the upstream DNA.
Following Translation by Single Ribosomes One Codon at a Time
In collaboration with Ignacio Tinoco Jr. (University of California, Berkeley), we are using a single-ribosome/single-mRNA helicase-based assay to characterize the dynamics of translation by single Escherichia coli ribosomes (see Figure 6). In these experiments, we follow individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. These studies have revealed that translation occurs through successive translocation-and-pause cycles (see Figure 7). 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 occurs in less than 0.1 seconds and measures three bases—one codon—indicating that translocation and RNA unwinding (i.e., the helicase activity of ribosomes) are strictly coupled ribosomal functions. Pause lengths, and therefore the overall rate of translation, depend on the secondary structure of the mRNA; force applied to the hairpin destabilizes its secondary structure and decreases pause durations but does not affect the translocation times.
As of May 30, 2012
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