Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented. The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263,17857-17871] and Rhee et al. [(1997) Biochemistry 36, 7664-7680], we find that the presence of an alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site. Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alphaL2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.

SUMMARY Recombinational repair of spontaneous double-strand breaks (DSBs) exhibits sister bias. DSB-initiated meiotic recombination exhibits homolog bias. Physical analysis in yeast reveals that, in both cases, recombination intrinsically gives homolog bias. From this baseline default, cohesin intervenes to confer sister bias, likely independent of cohesion. In meiosis, cohesin’s sister-biasing effect is counteracted by RecA-homolog Rad51 and its mediators, plus meiotic RecA- homolog Dmc1, which thereby restore intrinsic homolog bias. Meiotic axis complex Red1/Mek1/Hop1 participates by cleanly switching recombination from mitotic mode to meiotic mode, concomitantly activating Dmc1. We propose that a Rad51/DNA filament at one DSB end captures the intact sister, creating an “anchor pad”. This filament extends across the DSB site on the intact partner, precluding inter-sister strand exchange, thus forcing use of the homolog. Cohesin and Dmc1 interactively modulate this extension, giving program-appropriate effects. In accord with this model, Rad51-mediated recombination in vivo requires the presence of a sister.

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