Zinc finger proteins: A study of linker region importance, DNA, and RNA binding characteristics

Zinc fingers are a common structural motif found in nucleic acid binding proteins. Through interactions with nucleic acids they carry out many functions, including DNA binding to activate or repress RNA transcription and RNA binding for protection and shuttling of RNA throughout the cell. TFIIIA contains nine zinc fingers and binds the 5S RNA gene and its product, the 5S RNA. p43, a related nine zinc finger protein found in 42S RNP storage particles, also binds 5S RNA. Unlike TFIIIA, p43 is unable to bind the 5S RNA gene. The proteins share 33% sequence identity and common amino acids such as cysteines and histidines within the zinc fingers. The aim of this work has been to characterize the contribution to substrate binding strength and specificity by the zinc finger connecting region, the H/C link. Part of the highly conserved zinc finger H/C link region is TGE (Thr-Gly-Glu) and exists in H/C linkers one and two of TFIIIA. p43 does not contain TGE in any of its WC links. To characterize the potential function of these regions, amino terminal three zinc finger proteins from TFIIIA and p43 were made containing wild type or exchanged TFIIIA/p43 linker regions one and/or two. DNA and RNA dissociation constants were assessed with purified proteins. TFIIIA single and double mutant proteins can bind, but with a three to eight fold decrease in DNA binding affinity without significant change in RNA binding affinity. Additionally, exchanged linker regions substantially decreased DNA substrate specificity with a minor decrease in RNA specificity. Replacement of p43 H/C links with those from TFIIIA had no effect on RNA binding, however, this did not enable the TFIIIA linker-containing p43 protein to bind DNA. Evidently, DNA binding, and to a lesser extent RNA binding, is directly influenced by the H/C linker. Use of these three finger proteins has allowed determination of their binding position on 5S RNA for the first time. Coupling how and where zinc finger proteins interact with nucleic acids will enable us to design zinc finger proteins for therapeutic use