Kinetics of ATP/ADP binding to the gp16 ATPase.

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 µM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.

Anisotropy-Based Nucleosome Repositioning Assay.

Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction.

The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental processes within living cells, as well as holding great promise for the development of novel biomedical applications. Cell-free transcription-translation (TXTL) systems have become suitable platforms to tackle such topics because they recapitulate the process of gene expression. TXTL systems have advanced to where the in vitro construction of viable, complex, self-assembling deoxyribonucleic acid-programmed biological entities is now possible. Previously, we demonstrated the cell-free synthesis of three bacteriophages from their genomes: MS2, ΦX174, T7. In this work, we present the complete synthesis of the phage T4 from its 169-kbp genome in one-pot TXTL reactions. This achievement, for one of the largest coliphages, demonstrates the integration of complex gene regulation, metabolism and self-assembly, and brings the bottom-up synthesis of biological systems to a new level.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Low processivity for DNA translocation by the ISWI molecular motor.

The motor protein ISWI (Imitation SWItch) is the conserved catalytic ATPase domain of the ISWI family of chromatin remodelers. Members of the ISWI family are involved in regulating the structure of cellular chromatin during times of transcription, translation, and repair. Current models for the nucleosome repositioning activity of ISWI and other chromatin remodelers require the translocation of the remodeling protein along double-stranded DNA through an ATP-dependent mechanism. Here we report results from spectrofluorometric stopped-flow experiments which demonstrate that ISWI displays very low processivity for free DNA translocation. By combining these results with those from experiments monitoring the DNA stimulated ATPase activity of ISWI we further demonstrate that the DNA translocation by ISWI is tightly coupled to ATP hydrolysis. The calculated coupling efficiency of 0.067±0.018 ATP/ISWI/bp is seemingly quite low in comparison to similar DNA translocases and we present potential models to account for this. Nevertheless, the tight coupling of ATP hydrolysis to DNA translocation suggests that DNA translocation is not energetically rate limiting for nucleosome repositioning by ISWI.

ISWI remodels nucleosomes through a random walk.

The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.

Kinetic mechanism of DNA translocation by the RSC molecular motor.

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.