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Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40%o of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation. (2,5,26,30,46) and which remains functionally stable through many rounds of transcription (2,5,9,30,46 (1,13,20,32,33). It has also been suggested that a bend in the DNA template in the region of the termination signal may be important in termi-

The effects of a series of point mutations within the Xenopus borealis somatic-type 5S RNA gene on transcription factor IIIA (TFIIIA) binding affinity were quantified. These data define a critical sequence-dependent contact region within the classical box C promoter element from base pair 80 to 91. Substitution of GC base pairs at positions 81, 85, 86, 89, and 91 significantly reduce TFIIIA binding affinity. Base pairs located at other positions within the box C contact region provide a moderate contribution to TFIIIA-5S gene interaction. In contrast to the extensive set of sequence contacts within the box C element, TFIIIA interaction is localized primarily to two GC base pairs at positions 70 and 71 within the intermediate promoter element. A selected amplification and binding assay (SAAB) was performed with a synthetic internal control region (ICR) randomized from base pair 78 to 95 to identify box C promoter sequences bound with high affinity by TFIIIA. The wild-type 5S RNA gene sequence from 79 to 92 is strongly selected. These results are consistent with the critical role of the box C element in sequence-dependent promoter recognition by TFIIIA.

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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...

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Xenopus transcription factor IIIA (TFIIIA) binds to over 50 base pairs in the internal control region of the 5 S rRNA gene, yet the binding energy for this interaction (⌬G 0 ‫؍‬ ؊12.8 kcal/mol) is no greater than that exhibited by many proteins that occupy much smaller DNA targets. Despite considerable study, the distribution of the DNA binding energy among the various zinc fingers of TFIIIA remains poorly understood. By analyzing TFIIIA mutants with disruptions of individual zinc fingers, we have previously shown that each finger contributes favorably to binding (Del Rio, S., Menezes, S. R., and Setzer, D. R. (1993) J. Mol. Biol. 233, 567-579). Those results also suggested, however, that simultaneous binding by all nine zinc fingers of TFIIIA may involve a substantial energetic cost. Using complementary N-and C-terminal fragments and full-length proteins containing pairs of disrupted fingers, we now show that energetic interference indeed occurs between zinc fingers when TFIIIA binds to the 5 S rRNA gene and that the greatest interference occurs between fingers at opposite ends of the protein in the TFIIIA⅐5 S rRNA gene complex. Some, but not all, of the thermodynamically unfavorable strain in the TFIIIA⅐5 S rRNA gene complex may be derived from bending of the DNA that is necessary to accommodate simultaneous binding by all nine zinc fingers of TFIIIA. The energetics of DNA binding by TFIIIA thus emerges as a compromise between individual favorable contacts of importance along the length of the internal control region and long range strain or distortion in the protein, the 5 S rRNA gene, or both that is necessary to accommodate the various local interactions. Xenopus transcription factor IIIA (TFIIIA)1 is a multifunctional protein that recognizes the major cis-acting transcriptional control element in 5 S rRNA genes (the internal control region, or ICR) and thereby nucleates the formation of transcription complexes that result in the synthesis of 5 S rRNA (1-3). It also binds to 5 S rRNA itself to form a ribonucleoprotein storage particle that accumulates in Xenopus oocytes (4,5) and that may mediate feedback regulation of 5 S rRNA gene expression in somatic cells (5, 6). As the first sequence-specific DNA-binding protein to be purified from eukaryotic cells (1) and the archetypal zinc finger protein (7), TFIIIA has been an influential model protein for understanding the mechanisms of sequence-specific DNA and RNA recognition.The nine zinc fingers of TFIIIA define the sequence features that can be used to recognize zinc finger motifs in other proteins (7). These include two cysteine and two histidine residues with conserved spacing; these four amino acids coordinate a Zn 2ϩ ion (8) that contributes substantially to the stability of the folded domain (9 -11). Three conserved hydrophobic residues are also likely to stabilize TFIIIA-like zinc finger domains through interactions in a hydrophobic pocket. The large number of zinc fingers in TFIIIA has made its structural analysis difficult, and structural studies of ...