The role of zinc finger linkers in p43 and TFIIIA binding to 5S rRNA and DNA

Abstract

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…It has been previously observed that certain amino acid linker sequences connecting tandem repeats of C2H2 domains were highly correlated with ZF-DNA binding (Laity et al, 2000;Ryan and Darby, 1998). In mammals, tandem ZFs are frequently separated by linkers of 5 amino acids, with roughly 50% of these having the sequence TGEKP (Wolfe et al, 2000).…”

Reduction in DNA-binding affinity of Cys 2 His 2 zinc finger proteins by linker phosphorylation

“…Indeed, the conservation of this sequence allowed cDNAs encoding many zinc finger proteins to be identified before genomic sequencing efforts (3). Although structural studies (4-7) and mutagenesis experiments (8)(9)(10)(11)(12) have suggested a role for the linker in DNA binding, it has been noted that this contribution is relatively minor and probably insufficient to explain the high degree of evolutionary conservation of the linker sequence (10).…”

Structural Insights into c-Myc-interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding

“…However, as discussed elsewhere, the structural and dynamic characteristics dictating the functional role of ZFs are poorly understood (40). Tandems of ZFs are generally separated by highly conserved TGEKP sequence linkers that have been shown to be important for optimal DNA binding (18,19,42,43). Accordingly, different groups have proposed that the nature of ZF tandem linkers could enable the prediction of their functions (18,19,44).…”

“…Typically, series of ZFs are connected by highly conserved TGEKP sequence linkers and bind in sets of two or more in the major groove of the DNA with each ZF contacting three DNA bases. These linkers are important to allow for consecutive motifs to optimally fit in the major groove of DNA (18,19). Variations in the consensus sequence can significantly reduce binding affinity (18,19).…”

“…These linkers are important to allow for consecutive motifs to optimally fit in the major groove of DNA (18,19). Variations in the consensus sequence can significantly reduce binding affinity (18,19). Although not infallible, recognition codes and statistical potentials have been derived to predict the DNA sequences bound by ZF tandems from the nature of the residues at positions Ϫ1, 2, 3, and 6 of their recognition helix (17,20,21).…”