Functional interactions between the zinc fingers of Xenopus transcription factor IIIA during 5S rRNA binding.
We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.
Setzer
D R
DR
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA. drs9@po.cwru.edu
Menezes
S R
SR
Del Rio
S
S
Hung
V S
VS
Subramanyan
G
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D012259:Ribonuclease, Pancreatic