The sequence of the alternatively spliced sixth exon of alpha-tropomyosin is critical for cooperative actin binding but not for interaction with troponin.
Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D. (1991) BioEssays 13, 429-437). Exon 6, which encodes residues 189-213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution. In alpha-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms. Exon 6b encodes part of a putative Ca2+-sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions. A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function. Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase. The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin. The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present. However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence.
Hammell
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Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Hitchcock-DeGregori
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D000199:Actins / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers; D005091:Exons; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism; D014336:Troponin / Q000378:metabolism*
D000199:Actins; D017931:DNA Primers; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D002118:Calcium