Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries

Transcription factor IIIA (TFIIIA) binds to the 5S rRNA gene through its zi nc finger domain and directs the assembly of a multiprotein complex that pr omotes transcription initiation by RNA polymerase III. Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DN A binding and in vitro transcription analyses, have been used herein to inv estigate the domain organization and zinc requirements of Saccharomyces cer evisiae TFIIIA, Species containing either nine, six, or three zinc equivale nts were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA, Partial digestion of the metal-saturated, 9 Zn2+-lig anded factor yields a stable intermediate comprising the eight N-terminal z inc fingers, and a less stable fragment corresponding to a C-terminal porti on including the ninth finger. Proteolyzed TFIIIA has the same 5 S DNA bind ing ability of the intact protein yet no longer supports in vitro 5S rRNA s ynthesis. Both the structural compactness and the 5 S DNA binding ability o f the TFIIIA form only containing 3 zinc ions are severely compromised. In contrast, the 6 Zn2+-liganded species was found to be indistinguishable fro m metal-saturated TFIIIA. By demonstrating the existence of three classes o f zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexi bility built into this complex transcription factor.