RNA polymerase efficiently transcribes through DNA-scaffolded, cooperative bacteriophage repressor complexes.

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

Temperature-Dependent Nanomechanics and Topography of Bacteriophage T7.

Viruses are nanoscale infectious agents which may be inactivated by heat treatment. The global molecular mechanisms of virus inactivation and the thermally induced structural changes in viruses are not fully understood. In this study, we measured the heat-induced changes in the properties of T7 bacteriophage particles exposed to a two-stage (65°C and 80°C) thermal effect, by using atomic force microscopy (AFM)-based nanomechanical and topographical measurements. We found that exposure to 65°C led to the release of genomic DNA and to the loss of the capsid tail; hence, the T7 particles became destabilized. Further heating to 80°C surprisingly led to an increase in mechanical stability, due likely to partial denaturation of the capsomeric proteins kept within the global capsid arrangement.IMPORTANCE Even though the loss of DNA, caused by heat treatment, destabilizes the T7 phage, its capsid is remarkably able to withstand high temperatures with a more or less intact global topographical structure. Thus, partial denaturation within the global structural constraints of the viral capsid may have a stabilizing effect. Understanding the structural design of viruses may help in constructing artificial nanocapsules for the packaging and delivery of materials under harsh environmental conditions.

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch.

The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here we show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-microscope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol-1 at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.

Collective Force Regulation in Anti-parallel Microtubule Gliding by Dimeric Kif15 Kinesin Motors.

During cell division, the mitotic kinesin-5 Eg5 generates most of the force required to separate centrosomes during spindle assembly. However, Kif15, another mitotic kinesin, can replace Eg5 function, permitting mammalian cells to acquire resistance to Eg5 poisons. Unlike Eg5, the mechanism by which Kif15 generates centrosome separation forces is unknown. Here we investigated the mechanical properties and force generation capacity of Kif15 at the single-molecule level using optical tweezers. We found that the non-motor microtubule-binding tail domain interacts with the microtubule's E-hook tail with a rupture force higher than the stall force of the motor. This allows Kif15 dimers to productively and efficiently generate forces that could potentially slide microtubules apart. Using an in vitro optical trapping and fluorescence assay, we found that Kif15 slides anti-parallel microtubules apart with gradual force buildup while parallel microtubule bundles remain stationary with a small amount of antagonizing force generated. A stochastic simulation shows the essential role of Kif15's tail domain for load storage within the Kif15-microtubule system. These results suggest a mechanism for how Kif15 rescues bipolar spindle assembly.

Proteins mediating DNA loops effectively block transcription.

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

Stepwise reversible nanomechanical buckling in a viral capsid.

Viruses are nanoscale infectious agents constructed of a proteinaceous capsid that protects the packaged genomic material. Nanoindentation experiments using atomic force microscopy have, in recent years, provided unprecedented insight into the elastic properties, structural stability and maturation-dependent mechanical changes in viruses. However, the dynamics of capsid behavior are still unresolved. Here we used high-resolution nanoindentation experiments on mature, DNA-filled T7 bacteriophage particles. The elastic regime of the nanoindentation force trace contained discrete, stepwise transitions that cause buckling of the T7 capsid with magnitudes that are integer multiples of ∼0.6 nm. Remarkably, the transitions are reversible and contribute to the rapid consolidation of the capsid structure against a force during cantilever retraction. The stepwise transitions were present even following the removal of the genomic DNA by heat treatment, indicating that they are related to the structure and dynamics of the capsomeric proteins. Dynamic force spectroscopy experiments revealed that the thermally activated consolidation step is ∼104 times faster than spontaneous buckling, suggesting that the capsid stability is under strong dynamic control. Capsid structural dynamics may play an important role in protecting the genomic material from harsh environmental impacts. The nanomechanics approach employed here may be used to investigate the structural dynamics of other viruses and nanoscale containers as well.

[Current problems in the quality controll of pharmaceutical preparations manufactured in pharmacies I. Identification tests]. / Magisztrális gyógyszerkészítmények minoségellenorzésének aktuális kérdései I. Azonossági vizsgálatok.

Quality controll has fundamental importance in drug safety. During the last 15 years the regulatory changes resulted many problems in the quality controll of pharmaceutical preparations manufactured in pharmacys. The issue of Formulae Normales (FoNo VII.) and the 8th Hungarian Pharmacopoeia raises further questions. Our intentions to show the results have been achived in the area of elaborating new analytical prescriptions (identification and assay) which are crucial due to the obsolescence of previously used prescriptions.