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ABSTRACT: Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ∼4-fold. Stoichiometric considerations cause this activating effect to equate to an ∼47-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mamma- lian tropomyosin. To investigate these phenomena, chimeric tropomyosina were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM I. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPMI but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin SI increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-Ill. Similarly, myosin SI -ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM I - Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament. [ABSTRACT FROM AUTHOR]