Sulfolobus shibatae CCA-adding enzyme forms a tetramer upon binding two tRNA molecules: a scrunching-shuttling model of CCA specificity

The tRNA CCA-adding enzyme adds CCA stepwise to immature transfer RNA molecules untemplated, but with high specificity. We examined the oligomerization state of the enzyme from Sulfolobus shibatae and its binding to transfer RNA molecules, using various biophysical and biochemical methods including size exclusion chromatography, multi-angle laser light scattering, small-angle X-ray scattering, and gel electrophoresis band mobility shift assay. The 48 kDa monomer forms a stable salt- resistant dimer in solution. Further dimerization of the dimeric enzyme to form a tetramer is induced by the binding of two tRNA molecules. The formation of a tetramer with only two bound tRNA molecules leads us to suggest that one pair of active sites may be specific for adding two C bases, which results in scrunching of the primer strand. An adjacent second pair of active sites may be specific for adding A after addition of two C bases which makes the 3′ terminus long enough to reach the second pair of active sites.