Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage 29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for 29, averaging Ϸ700 bp/s and ranging up to Ϸ2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in <10 min during viral infection and implying high ATP turnover rates of >300 s ؊1 . The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.bacteriophage T4 ͉ molecular motor ͉ optical tweezers ͉ single-molecule ͉ viral DNA packaging A critical step in the assembly of many viruses is the packaging of the viral genome into a preassembled prohead shell by the action of an ATP-powered molecular motor (1, 2). Systems in which this mode of assembly occurs include numerous tailed dsDNA and dsRNA phages and certain animal viruses, including adenoviruses and herpesviruses. Viral DNA packaging complexes thus constitute a wide and potentially diverse family of molecular motors that are considerably understudied compared with cellular molecular motors such as myosins, kinesins, and helicases.In a typical phage assembly pathway, a prohead shell of precise dimensions co-assembles with a scaffolding core. One of the vertices of the prohead is unique, containing a dodecameric portal ring structure (3). When the scaffolding leaves, a defined space is created inside the capsid. A packaging ATPase complex then docks onto the outer end of the portal, inserting one end of the viral genome into the 3.5-to 4-nm channel, and translocates the DNA by using ATP hydrolysis energy (2, 4). After genome packaging, the ATPase dissociates, leaving the portal with the head, the outer surface of which provides a platform for the assembly of tail components. When the virus infects a cell, the densely packed DNA exits rapidly through the portal ch...

Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

It is well known that a jostled string tends to become knotted; yet the factors governing the ''spontaneous'' formation of various knots are unclear. We performed experiments in which a string was tumbled inside a box and found that complex knots often form within seconds. We used mathematical knot theory to analyze the knots. Above a critical string length, the probability P of knotting at first increased sharply with length but then saturated below 100%. This behavior differs from that of mathematical self-avoiding random walks, where P has been proven to approach 100%. Finite agitation time and jamming of the string due to its stiffness result in lower probability, but P approaches 100% with long, flexible strings. We analyzed the knots by calculating their Jones polynomials via computer analysis of digital photos of the string. Remarkably, almost all were identified as prime knots: 120 different types, having minimum crossing numbers up to 11, were observed in 3,415 trials. All prime knots with up to seven crossings were observed. The relative probability of forming a knot decreased exponentially with minimum crossing number and Mö bius energy, mathematical measures of knot complexity. Based on the observation that long, stiff strings tend to form a coiled structure when confined, we propose a simple model to describe the knot formation based on random ''braid moves'' of the string end. Our model can qualitatively account for the observed distribution of knots and dependence on agitation time and string length.Jones polynomial ͉ knot energy ͉ knot theory ͉ random walk ͉ statistical physics K nots have been a subject of scientific study since as early as 1867, when Lord Kelvin proposed that atoms might be described as knots of swirling vortices (1). Although this theory fell into disfavor, it stimulated interest in the subject, and knots currently play a role in many scientific fields, including polymer physics, statistical mechanics, quantum field theory, and DNA biochemistry (2, 3). Knotting and unknotting of DNA molecules occurs in living cells and viruses and has been extensively studied by molecular biologists (4-6). In physics, spontaneous knotting and unknotting of vibrated ball-chains have recently been studied (7-9). In mathematics, knot theory has been an active field of research for more than a century (3).Formation of knots in mathematical self-avoiding random walks has been extensively studied (10-16). In the 1960s, Frisch and Wasserman (10) and Delbruck (11) conjectured that the probability of finding a knot would approach 100% with an increasing walk length. In 1988, Sumners and Whittington (15) proved this conjecture rigorously by showing that exponentially few arcs would remain unknotted as the length tends to infinity. Numerical studies of finite-length random walks find that the probability of knotting and the average complexity of knots increase sharply with the number of steps (16).Here, we describe a simple physical experiment on knot formation. A string was placed in a cubic box and the...

Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels.

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20× higher force than generated by the skeletal muscle myosin II motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit differences in DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.