fluorescence ͉ microscopy ͉ virus ͉ genome T he transfer of bacteriophage DNA from a capsid into the host cell is an event of great importance to biology and physics. In biology, DNA ejection was a key piece of evidence demonstrating that the genetic material was DNA and not protein (1), phages have long been used to insert foreign genes into bacteria (2), and phage-mediated DNA transfer between species is a challenge to theories of evolution (3). In physics, the translocation of DNA through a pore has been studied from the theoretical and experimental points of view (4-8). Because phage DNA ejection is such a well known example of this process, it is important to understand it from a quantitative point of view. This paper addresses a longstanding, quantitative puzzle about phage DNA ejection: How fast is the ejection process? We use bacteriophage , a typical tailed phage, to answer this question. In a infection, first the phage tail binds to the Escherichia coli outer membrane protein LamB, triggering ejection. Then the genome, 48.5 kbp of double-stranded DNA, moves out of the phage head, through the tail, and into the cytoplasmic space, which requires force on the DNA directed into the cell. A force of tens of piconewtons (pN) is produced by the highly bent and compressed DNA within the capsid (9-11), but not much is known about how fast the DNA transfer occurs, except that ejection reaches completion in vivo in Ͻ2 min (12). One study used lipid vesicles incorporating LamB and filled with ethidium bromide: the DNA was ejected into the vesicles, causing an increase in fluorescence over Ϸ30 s (13). However, the Ϸ1,000 molecules of ethidium bromide in each vesicle were enough for only the first 1 kbp of DNA (14). Also, because the ejections could have started at different times, that experiment says very little about the DNA translocation process. This paper aims to resolve these challenges in describing the ejection process.An important insight from theory is that frictional forces limit the speed of ejection, due to DNA rearrangement in the phage head or sliding forces in the tail (15,16). Because the DNA is in a liquid state (17), we expect friction to behave at least somewhat like macroscopic hydrodynamic drag: stronger at higher speed or at smaller spacings between the moving parts. The DNA-tail interaction does not change during the ejection process, so we expect friction in the tail to remain constant. In contrast, friction in the head should be stronger when the spacing between the loops of DNA is small, i.e., at the beginning of ejection.To quantify the rate of ejection, a single-phage technique is necessary. Single-phage ejections were first observed with fluorescence microscopy on phage T5, revealing an effect of the unique structure of the T5 genome: nicks in the DNA resulted in predefined stopping points and a stepwise translocation process, with speeds that were too high to be quantified, so that further analysis of the speed and source of friction was not possible (18). As we will show here, ejects...
The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes) in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.
We present an exquisite 30 minute cadence Kepler (K2) light curve of the Type Ia supernova (SN Ia) 2018oh (ASASSN-18bt), starting weeks before explosion, covering the moment of explosion and the subsequent rise, and continuing past peak brightness. These data are supplemented by multi-color Panoramic Survey Telescope (Pan-STARRS1) and Rapid Response System 1 and Cerro Tololo Inter-American Observatory 4 m Dark Energy Camera (CTIO 4-m DECam) observations obtained within hours of explosion. The K2 light curve has an unusual twocomponent shape, where the flux rises with a steep linear gradient for the first few days, followed by a quadratic rise as seen for typical supernovae (SNe)Ia. This "flux excess" relative to canonical SNIa behavior is confirmed in our i-band light curve, and furthermore, SN 2018oh is especially blue during the early epochs. The flux excess peaks 2.14±0.04 days after explosion, has a FWHM of 3.12±0.04 days, a blackbody temperature of T 17, 500 9,000 11,500 =-+ K, a peak luminosity of 4.3 0.2 10 erg s 37 1 ´-, and a total integrated energy of 1.27 0.01 10 erg 43 ´. We compare SN 2018oh to several models that may provide additional heating at early times, including collision with a companion and a shallow concentration of radioactive nickel. While all of these models generally reproduce the early K2 light curve shape, we slightly favor a companion interaction, at a distance of ∼2 10 cm 12 based on our early color measurements, although the exact distance depends on the uncertain viewing angle. Additional confirmation of a companion interaction in future modeling and observations of SN 2018oh would provide strong support for a single-degenerate progenitor system.
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