A feedback loop coupling 5 S rRNA synthesis to accumulation of a ribosomal protein.

We have shown that elevated expression of ribosomal protein L5 in Xenopus embryos results in the ectopic activation of 5 S rRNA genes that are normally inactive. This transcriptional stimulation mimics the effect of overexpressing transcription factor IIIA (TFIIIA), the 5 S rRNA gene-specific transcription factor. The results support a model in which a network of nucleic acid-protein interactions involving 5 S rRNA, the 5 S rRNA gene, TFIIIA, and L5 mediates both feedback inhibition of 5 S rRNA synthesis and coupling of 5 S rRNA synthesis to accumulation of a ribosomal protein, L5. We propose that these mechanisms contribute to the homeostatic control of ribosome assembly.

Genetic analysis of Xenopus transcription factor IIIA.

We describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA). In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences. This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity. We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity. Over 30 such mutants have been identified and partially characterized. The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene. Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available. Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast.

Energetically unfavorable interactions among the zinc fingers of transcription factor IIIA when bound to the 5 S rRNA gene.

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 (DeltaG0 = -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 TFIIIA.5 S rRNA gene complex. Some, but not all, of the thermodynamically unfavorable strain in the TFIIIA.5 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.

Functional interactions between the zinc fingers of Xenopus transcription factor IIIA during 5S rRNA binding.

We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.

Contribution of individual base pairs to the interaction of TFIIIA with the Xenopus 5S RNA gene.

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.

The function of individual zinc fingers in sequence-specific DNA recognition by transcription factor IIIA.

Mutations in Zn(2+)-coordinating histidine residues have been used to generate forms of transcription factor IIIA containing structural disruptions in single zinc fingers. These mutant proteins have been analyzed with respect to the structural and functional independence of individual zinc fingers in TFIIIA. They have also been used to assess the energetic contributions to binding and the sites of interaction of each of the nine zinc fingers of TFIIIA with the 5 S rRNA gene. The results are surprising and suggest a complex mode of binding in which the interactions of structurally independent zinc fingers with the DNA substrate are non-uniform and functionally interdependent in a way that may help to explain some of the unusual properties of TFIIIA.

Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

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

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.

Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition.

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% 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.

High yield purification of active transcription factor IIIA expressed in E. coli.

Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture. This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA). Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source. Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes. We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.

Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase.

In the absence of other components of the RNA polymerase III transcription machinery, transcription factor IIIA (TFIIIA) can be displaced from both strands of its DNA-binding site (the internal control region) on the somatic-type 5S rRNA gene of Xenopus borealis during transcription elongation by bacteriophage T7 RNA polymerase, regardless of which DNA strand is transcribed. Furthermore, substantial displacement is observed after the template has been transcribed only once. Since the complete 5S rRNA transcription complex has previously been shown to remain stably bound to the gene during repeated rounds of transcription by either RNA polymerase III or bacteriophage SP6 RNA polymerase, these results indicate that a factor(s) in addition to TFIIIA is required to create a complex that will remain stably associated with the template during transcription. Thus, transcription complex stability during passage of RNA polymerase cannot be explained solely on the basis of the DNA-binding properties of TFIIIA.

A simple vector modification to facilitate oligonucleotide-directed mutagenesis.

We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.

Formation and stability of the 5 S RNA transcription complex.

5 S ribosomal RNA in Xenopus has been shown to be transcribed in vitro from 5 S RNA genes that remain stably associated with required transcription factors through multiple rounds of transcription (Bogenhagen, D. F., Wormington, W. M., and Brown, D. D. (1982) Cell 28, 413-421). We have studied the formation and stability of these "transcription complexes" by using cloned 5 S RNA genes immobilized on cellulose as templates for the assembly of complexes in crude extracts. RNA polymerase III is the least tightly bound component required for transcription of 5 S RNA genes. All other factors remain bound in 1 M NaCl, even though transcription complexes do not form at salt concentrations as low as 0.25 M. RNA polymerase III dissociates from transcription complexes as a result of RNA synthesis and is capable of reassociating with complexes to support additional rounds of transcription. A 5 S-specific positive transcription factor (factor A) and two crude phosphocellulose column fractions (B and C) are also required for 5 S RNA synthesis in vitro (Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980) Cell 19, 717-728; Segall, J., Matsui, T., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11986-11991; Shastry, B. S., Ng, S.-Y., and Roeder, R. G. (1982) J. Biol. Chem. 257, 12979-12986). Fraction B stably interacts with 5 S RNA genes to form a stable, active complex only after the template has first been incubated with factor A and fraction C. In contrast, either factor A or fraction C can stably associate with 5 S RNA genes in the absence of other factors. The activities of fractions B and C are removed from solution as a result of transcription complex formation, suggesting the factors in these fractions act stoichiometrically. The rate-limiting step in complex formation is carried out by fraction B, which accounts for the lag in transcription activity observed in crude extracts.

A family of repeated DNA sequences, one of which resides in the second intervening sequence of the mouse dihydrofolate reductase gene.

One member of a repeated sequence family has been found in the second intron of the mouse dihydrofolate reductase gene. The family consists of approximately 20 members/haploid genome, two of which have been sequenced and found to contain a 480-base pair region of homology with a significant amount of sequence divergence. Rat, Chinese hamster, Syrian hamster, and human DNAs contain homologous repetitious elements, and the four rodent species have a member of this family associated with the dihydrofolate reductase gene.

Nucleotide sequence surrounding multiple polyadenylation sites in the mouse dihydrofolate reductase gene.

We have previously reported the presence of four dihydrofolate reductase messenger RNAs differing in the length of 3' untranslated regions in murine cells (Setzer, D. R., McGrogan, M., Nunberg, J. H., and Schimke, R. T. (1980) Cell 22, 361-370). We have now mapped the 3' ends of these RNAs more precisely and have demonstrated colinearity between their shared sequences. Analysis of three larger dihydrofolate reductase RNAs has shown that these RNA species contain very long 3' noncoding regions, bringing the total number of dihydrofolate reductase RNAs to seven, ranging in length from 750 to 5600 nucleotides. We have determined the nucleotide sequence at and surrounding the polyadenylation sites of the four smaller RNAs. We find no striking structures in this sequence that might constitute multiple polyadenylation signals, but conclude that the putative polyadenylation signal AAUAAA is not required for polyadenylation of at least three of the four dihydrofolate reductase messengers.

Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells.

We have examined in detail the RNA coding for dihydrofolate reductase (DHFR) in methotrexate-resistant mouse cells. We find four distinct DHFR messengers, ranging in size from 750 to 1600 nucleotides. All four are polyadenylated and polysomal and can be translated in vitro to produce a 21,000 dalton protein co-migrating with purified dihydrofolate reductase on SDS polyacrylamide gels. The major difference in these RNAs is the length of 3' untranslated regions, varying from about 80 nucleotides in the smallest mRNA to about 930 nucleotides in the largest. The RNAs are also present in methotrexate-sensitive murine cells and mouse liver. Multiple DHFR RNAs are found in the poly(A)+ RNA of methotrexate-resistant Chinese hamster ovary cells but are of different molecular weights than the mouse messengers. We discuss these results in terms of the function of 3' untranslated regions of eucaryotic mRNAs and the possible origin and significance of multiple messenger RNAs for a single protein.