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...

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Packaged viral genome can be removed from bacteriophage T4 capsid and the capsid refilled with any double-stranded DNA, single or multiple molecules, using a powerful ATP-fueled DNA packaging machine.

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ∼170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an offtranslocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.virus assembly | DNA translocase | optical trap T ailed bacteriophages are considered to be the most abundant forms of life on earth (1). In late stages of their life cycle, the newly replicated viral genome is translocated into a preformed "head" through a special portal vertex and compacted to near crystalline density (∼500 mg/mL) (2). Powerful molecular motors are used to drive this process, which can generate forces >80 pN to overcome bending and repulsive forces that resist DNA confinement and compaction (3). The phage T4 motor, which packages at a rate of up to ∼2,000 bp/s and has a power density of ∼5,000 kW/m 3 , is one of the fastest and most powerful molecular motors known (4). The T4 motor is also one of the best studied viral packaging machines, with extensive biochemical characterizations coupled with genetic analyses, atomic structures, and structural modeling (5, 6). It is a prototype for the packaging motors of phages and herpes viruses and belongs to the diverse additional strand, conserved E (ASCE) superfamily of homooligomeric motors, which includes hexameric helicases, protein translocases, and type III restriction enzymes (7).The T4 packaging motor is a pentamer of the large terminase protein, gp17 (70 kDa), assembled at the portal vertex of the empty prohead (Fig. 1A) (6, 8, 9). It is a key component of the packaging machine that consists of two additional components: the dodecameric portal (61 kDa gp20) that provides the ∼35 Å central channel through which DNA enters ...