Identification and characterization of transcription factor IIIA from Schizosaccharomyces pombe

Abstract

Transcription factor IIIA (TFIIIA) is specifically required for transcription of 5S rRNA genes and is the archetypal C2H2 zinc finger protein. All known vertebrate TFIIIAs have a similar organization: nine zinc fingers, followed by a C-terminal domain of unknown structure. The zinc fingers of Saccharomyces cerevisiae TFIIIA are interrupted between fingers eight and nine by an 81-amino acid spacer. Aside from the amino acids required for zinc finger folding, TFIIIAs from different species are remarkably divergent, whereas the natural binding site, the internal control region of the 5S rRNA gene, is well conserved. We now describe the identification and characterization of TFIIIA from Schizosaccharomyces pombe. This protein is organized differently from its known homologs, in that it contains eight closely spaced zinc fingers, a ninth zinc finger missing a C-terminal Zn2+-coordinating histidine, a 53- amino acid spacer, and an unprecedented tenth zinc finger. We have confirmed the identity of this divergent protein as TFIIIA by showing that it binds specifically and with high affinity to the S.pombe 5S rRNA gene. Comparison of DNase I protection patterns produced by TFIIIA from multiple species suggests a novel mode of DNA recognition by the S.pombe protein. Recombinant S.pombe TFIIIA was also shown to support specific transcription of the 5S rRNA gene in vitro.

Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches

“…A number of different techniques have been developed for purification of TFs, which is an important step prior to studies on the structure and functions of TFs (Jiang et al, 2007;Maouche and Cohen-Kaminsky, 1997;Schulman and Setzer, 2002;Yang, 1998). In principle, these methods are all based on the ability of TFs to recognize and interact with specific DNA sequences present in the promoters of eukaryotic genes.…”

Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

“…The ninth zinc finger of the recently characterized TFIIIA from Schizosaccharomyces pombe , which contains an unprecedented 10 potential zinc fingers, lacks the carboxyl-terminal Zn 2+ -coordinating histidine residue. This module is nonetheless able to fold and function in DNA binding and TF activity (10,55). Based on studies with synthetic, truncated zinc -finger polypeptides that showed that these peptides are able to fold in the absence of a carboxyl-terminal histidine residue (56), Schulman and Setzer (2003) speculate that a water molecule or a dithiothreitol-provided thiolate acts as the fourth zinc ligand in finger 9 of the S. pombe homolog of TFIIIA.…”

“…However, TFIIIAs with longer spacer regions and additional zinc fingers have been identified in some species (9,10). The interaction of TFIIIA from Xenopus with the tripartite ICR of the amphibian 5 S RNA gene has been studied in detail.…”

Hybrid Modeling of Noise Reduction by a Negatively Autoregulated System

“…In the literature, the following orders of magnitude for the different rates are stated. Values for the turning on and off rates are reported in the order of 10 −3 min −1 , and 10 −3 to 10 −1 min −1 , respectively (Kwon et al, 2001;Schulman and Setzera, 2002). A realistic value for the production rate lies in the order of 1 to 10 nM min −1 (Sørensen and Pedersen, 1991).…”