ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats

Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.     

Do cells think?

A microorganism has to adapt to changing environmental conditions in order to survive. Cells could follow one of two basic strategies to address such environmental fluctuations. On the one hand, cells could anticipate a fluctuating environment by spontaneously generating a phenotypically diverse population of cells, with each subpopulation exhibiting different capacities to flourish in the different conditions. Alternatively, cells could sense changes in the surrounding conditions – such as temperature, nutritional availability or the presence of other individuals – and modify their behavior to provide an appropriate response to that information. As we describe, examples of both strategies abound among different microorganisms. Moreover, successful application of either strategy requires a level of memory and information processing that has not been normally associated with single cells, suggesting that such organisms do in fact have the capacity to ‘think’. Received 3 January 2007; accepted 4 April 2007     

Cross-talk and decision making in MAP kinase pathways

Cells must respond specifically to different environmental stimuli in order to survive. The signal transduction pathways involved in sensing these stimuli often share the same or homologous proteins. Despite potential cross-wiring, cells show specificity of response. We show, through modeling, that the physiological response of such pathways exposed to simultaneous and temporally ordered inputs can demonstrate system-level mechanisms by which pathways achieve specificity. We apply these results to the hyperosmolar and pheromone mitogen-activated protein (MAP) kinase pathways in the yeast Saccharomyces cerevisiae. These two pathways specifically sense osmolar and pheromone signals, despite sharing a MAPKKK, Ste11, and having homologous MAPKs (Fus3 and Hog1). We show that in a single cell, the pathways are bistable over a range of inputs, and the cell responds to only one stimulus even when exposed to both. Our results imply that these pathways achieve specificity by filtering out spurious cross-talk through mutual inhibition. The variability between cells allows for heterogeneity of the decisions.