The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.

The recA protein catalyses the ATP-driven homologous pairing and strand exchange of DNA molecules. It is an allosteric enzyme: the ATPase activity is DNA-dependent, and ATP-bound recA protein has a high affinity for DNA, whereas the ADP-bound form has a low affinity. In the absence of ATP hydrolysis, recA protein can still promote homologous pairing, apparently through the formation of a triple-stranded intermediate. The exact role of ATP hydrolysis is not clear, but it presumably drives the triplex intermediate towards products. Here we determine the position of bound ADP diffused into the recA crystal. We show that only the phosphates are bound in the same way as in other NTPases containing the G/AXXXXGKT/S motif. We propose that recA protein may change its conformation upon ATP hydrolysis in a manner analogous to one such protein, the p21 protein from the ras oncogene. A model is presented to account for the allosteric stimulation of DNA binding by ATP. The mechanism by which nucleoside triphosphate hydrolysis is coupled to the binding of another ligand in recA protein and p21 may be typical of the large class of NTPases containing this conserved motif.

was an error in the algorithm used to generate the 2-13 C and 4-13 C glutamate turnover curves. Consequently, we recalculated the tricarboxylic acid (TCA) cycle flux by using CWAVE software (Graeme F. Mason, Yale University, New Haven, CT). This mathematical modeling was based on nonlinear least squares fitting of the calculated parameters (4-and 2-13 C citrate, ␣-ketoglutarate, glutamate) from the set of isotopic mass balance equations describing the label flow through the TCA cycle to the acquired NMR data using a Runge-Kutta algorithm with an adaptive step size. After recalculation, the absolute TCA cycle flux in all groups [control, triido-L-thyronine (T 3 ), and 2,4-dinitrophenol (DNP)] was found to be higher than originally reported. Consistent with our initial estimates, the revised calculations indicate that the TCA cycle flux (Fig. 3B) did significantly increase in the T 3 and DNP groups vs. the control group (P Ͻ 0.05 and P Ͻ 0.01, respectively). Additionally, the mitochondrial energy coupling (Fig. 3C) was reduced in the T 3 and DNP groups vs. the control group (P Ͻ 0.01 and P Ͻ 0.001, respectively) with no significant difference between the T 3 and DNP groups. Therefore, although the absolute TCA cycle fluxes have increased as a result of the new calculations, our main conclusion regarding a reduction in mitochondrial energy coupling following T 3 and DNP treatments remains unchanged. Fig. 3 shows the results of our revised calculations.www.pnas.org͞cgi͞doi͞10.1073͞pnas.081073598 (A and B) The calculated unidirectional ATP synthesis (A) and TCA cycle flux (B). There was a significant increase in TCA cycle flux in the T 3-treated vs. the control group and a much greater increase in the DNP-treated rats. These data suggest that, although tissue viability was not compromised by the T 3 and DNP treatment as reflected by similar ATP synthesis flux in all three groups (A), there was a significantly increased rate of substrate oxidation required to generate the ATP (B). Therefore, when comparing the degree of coupling (normalized ratio of ATP synthesis flux to TCA cycle flux) between these two measurements (C), it is evident that there was significantly greater mitochondrial uncoupling occurring in the T 3 and DNP vs. control group. Data are presented as means Ϯ SEM. N.S., not significant.