Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries.
Transcription factor IIIA (TFIIIA) binds to the 5 S rRNA gene through its zinc finger domain and directs the assembly of a multiprotein complex that promotes transcription initiation by RNA polymerase III. Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DNA binding and in vitro transcription analyses, have been used herein to investigate the domain organization and zinc requirements of Saccharomyces cerevisiae TFIIIA. Species containing either nine, six, or three zinc equivalents were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA. Partial digestion of the metal-saturated, 9 Zn2+-liganded factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a C-terminal portion including the ninth finger. Proteolyzed TFIIIA has the same 5 S DNA binding ability of the intact protein yet no longer supports in vitro 5 S rRNA synthesis. Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only containing 3 zinc ions are severely compromised. In contrast, the 6 Zn2+-liganded species was found to be indistinguishable from metal-saturated TFIIIA. By demonstrating the existence of three classes of zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexibility built into this complex transcription factor.
Pizzi
S
S
Institute of Biochemical Sciences, University of Parma, I-43100 Parma, Italy.
Dieci
G
G
Frigeri
P
P
Piccoli
G
G
Stocchi
V
V
Ottonello
S
S
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D015032:Zinc