Analysis of the molecular mechanisms for the species-specific transcription of Drosophila and human tRNA gene transcription components.

The transcription of eucaryotic tRNA genes requires two factors IIIB and IIIC, in addition to RNA polymerase III, to reconstitute this process in vitro. We have examined the functional exchangeability of these components from Drosophila and human systems. The reconstitution of heterologous IIIB and IIIC components demonstrated that neither factor will functionally substitute for the homologous components to activate tRNA gene transcription. The addition of the heterologous Drosophila factors to HeLa transcription assays causes an inhibition of RNA synthesis that is dependent upon the order of addition of these proteins to the DNA template. Thus, it appears that tRNA gene transcription in these systems is species-specific. We have further analyzed the reason for the apparent incompatibilities of these components by the use of stable complex formation assays. We find that human HeLa IIIB and Drosophila IIIC are unable to form stably associated complexes with a tRNA gene template, whereas the Drosophila IIIB and HeLa IIIC do form stable but nonproductive complexes. These results demonstrate that specific IIIC-IIIB interactions are critical in the formation of productive transcription complexes and are responsible for the observed species specificity of Drosophila and human tRNA gene transcription.