The role of zinc fingers in transcriptional activation by transcription factor IIIA.

Abstract

We have described elsewhere a number of the properties of a set of mutant forms of Xenopus transcription factor IIIA (TFIIIA) containing single amino acid substitutions that result in the structural disruption of individual zinc finger domains. These "broken finger" proteins have now been analyzed with respect to their ability to support transcription of 5S rRNA genes in vitro. Disruption of any one of the first six zinc fingers of TFIIIA has no discernible effect on the activity of the protein in supporting 5S rRNA synthesis in standard in vitro transcription assays, despite the fact that some of these mutant proteins exhibit large decreases in their binding affinity for 5S rRNA genes in binary complexes. These results indicate that the activity of TFIIIA as a transcription factor can be largely independent of its equilibrium binding constant for the 5S rRNA gene in the absence of other components of the RNA polymerase III transcriptional apparatus. In fact, this finding is consistent with the known pathway and kinetics of assembly of 5S rRNA transcription complexes. In contrast to the results obtained with finger 1-6 mutants, analogous mutations in zinc fingers 7-9 of TFIIIA result in moderate to complete loss of transcriptional activity. We interpret these results to mean that the three C-terminal zinc fingers of TFIIIA are not only involved in binding to the internal control region of 5S rRNA genes but are also required, either directly or indirectly, for higher-order interactions that are important in transcription complex assembly, stability, or activity.Since the landmark experiments of Brent and Ptashne (1), the modular structure of sequence-specific eukaryotic transcription factors has become a central feature of most models of the eukaryotic transcriptional apparatus. This point of view has been repeatedly validated by the ability to structurally and functionally define discrete and independent domains for DNA binding and transcriptional activation in many transcription factors. Transcriptional activation domains are generally presumed to be sites of interaction with other proteins and capable of acting to promote transcription initiation when fused to virtually any DNA-binding domain. While most of these studies have been carried out with sequence-specific class II factors-that is, those that activate transcription by RNA polymerase II-some data suggest that functionally analogous domains exist in the class I factor UBF (2) and in the 5S rRNA gene-specific class III factor transcription factor IIIA (TFIIIA) (3). In the case of TFIIIA, =:z75% of the protein consists of the nine consecutive zinc finger domains (4) shown to contain the DNA-binding activity of the factor, while a shorter sequence near the C terminus of TFIIIA has been demonstrated to be required for transcriptional activation (3).Despite the apparent separation of DNA-binding and transcriptional activation functions in most transcription factors, it is often implicitly assumed that mutations that reduce DNA-binding affinity will r...

Energetically Unfavorable Interactions among the Zinc Fingers of Transcription Factor IIIA When Bound to the 5 S rRNA Gene

“…Our observation of compensatory binding and energetic buffering, combined with our previous report on the insensitivity of TFIIIA function in in vitro transcription assays to the binding affinity of TFIIIA in binary complexes with the 5 S rRNA gene (63), may provide an explanation for this lack of sequence conservation. We suggest that mutational alterations in TFIIIA would have relatively modest effects on DNA binding affinity and that the ultimate effects of these changes on the transcriptional function of TFIIIA would be further mitigated by the tremendous stabilization of binding that is afforded by the subsequent association of TFIIIC and TFIIIB.…”

Two Types of Zinc Fingers Are Required for Dimerization of the serendipity δ Transcriptional Activator

“…The crystal structure of the five-finger Gli protein-DNA complex showed that, in other subclasses, separate zinc fingers can contribute unequally to DNA recognition (26). Furthermore, some Cys 2 /His 2 zinc fingers have been shown to mediate protein contacts (7,21), subcellular localization (19), or a puzzling nuclease activity (12). Therefore, for a given protein, the role of individual Cys 2 /His 2 fingers in both DNA binding and protein-protein contacts remains an open question.…”

A position-dependent transcription-activating domain in TFIIIA.

“…Protein-protein interactions with the zinc fingers could account for this stabilization. A role for zinc fingers in transcriptional activation distinct from DNA binding has been suggested (13). Complex stability is not a prerequisite for transcription (51), so differences in stability of complexes formed with C-terminal mutants are not necessarily reflected by changes in transcriptional activation.…”

“…Carboxyl-terminal zinc fingers may also be required for transcriptional activation in addition to DNA binding. Disruptions of single zinc fingers in the C-terminal portion of the zinc finger domain, made by mutation of a single histidine to asparagine, result in reduced transcription without changing the affinity of TFIIIA for the ICR (13).…”

“…In the first PCR, forward primers complementary to the 3' limit of the desired deletion (BRDP, FDIP, and FDIIP, respectively) were used to generate an NgoMI-HindIII frag-VOL. 13,1993 7498 MAO AND DARBY ment. This fragment was ligated to the XhoI-NgoMI fragment from the second PCR, using primers P673 and ZF9RP, and subcloned into pMSTF as described above.…”