Promoter-specific shifts in transcription initiation conferred by yeast TFIIB mutations are determined by the sequence in the immediate vicinity of the start sites.

The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB.

The effect of bone morphogenetic protein-7 on the expression of type I inositol 1,4,5-trisphosphate receptor in G-292 osteosarcoma cells and primary osteoblast cultures.

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

An interaction between the N-terminal region and the core domain of yeast TFIIB promotes the formation of TATA-binding protein-TFIIB-DNA complexes.

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by eukaryotic RNA polymerase II. We previously identified a yeast TFIIB mutant (R64E) that exhibited increased activity in the formation of stable TATA-binding protein-TFIIB-DNA (DB) complexes in vitro. We report here that the homologous human TFIIB mutant (R53E) also displayed increased activity in DB complex formation in vitro. Biochemical analyses revealed that the increased activity of the R64E mutant in DB complex formation was associated with an altered protease sensitivity of the protein and an enhanced interaction between the N-terminal region and the C-terminal core domain. These results suggest that the intramolecular interaction in yeast TFIIB stabilizes a productive conformation of the protein for the association with promoter-bound TATA-binding protein.

The N-terminal region of yeast TFIIB contains two adjacent functional domains involved in stable RNA polymerase II binding and transcription start site selection.

The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro. Mutations impairing the formation of stable TFIIB.RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB.RNAPII binding. We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase.

Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes.

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.

The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae.

Eukaryotic transcriptional activators have been classified on the basis of the characteristics of their activation domains. Acidic activation domains, such as those in the yeast GAL4 or GNC4 proteins and the herpes simplex virus activator VP16, stimulate RNA polymerase II transcription when introduced into a variety of eukaryotic cells. This species interchangeability demonstrates that the mechanism by which acidic activation domains function is highly conserved in the eukaryotic kingdom. To determine whether such a conservation of function exists for a different class of activation domain, we have tested whether the glutamine-rich activation domains of the human transcriptional activator Sp1 function in the yeast Saccharomyces cerevisiae. We report here that the glutamine-rich domains of Sp1 do not stimulate transcription in S. cerevisiae, even when accompanied by human TATA-box binding protein (TBP) or human-yeast TATA-box binding protein hybrids. Thus, in contrast to the case for acidic activation domains, the mechanism by which glutamine-rich domains stimulate transcription is not conserved between S. cerevisiae and humans.

Yeast and human TFIIDs are interchangeable for the response to acidic transcriptional activators in vitro.

Previous work showed that human TFIID fails to support yeast cell growth, although it is nearly identical to yeast TFIID in a carboxy-terminal region of the molecule that suffices for basal, TATA-element-dependent transcription in vitro. These and other findings raised the possibility that TFIID participates in species-specific interactions, possibly with mediator factors, required for activated transcription. Here, we report that human TFIID and amino-terminally truncated derivatives of yeast TFIID are fully functional in support of both basal transcription and the response to acidic activator proteins in a yeast in vitro transcription system. Conversely, and in contrast to previously published results, yeast TFIID supports both basal and activated transcription in reactions reconstituted with human components. This functional interchangeability of yeast and human TFIIDs argues strongly against species specificity with regard to TFIID function in basal transcription and the response to acidic activator proteins. In addition, our results suggest that any intermediary factors between acidic activators and TFIID are conserved from yeast to man.

Chromosomal context dependence of a eukaryotic recombinational hot spot.

The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting.

Functional differences between yeast and human TFIID are localized to the highly conserved region.

TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Meiotic recombination-deficient mutants of Schizosaccharomyces pombe.

A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs. intergenic) specificity of the corresponding rec+ gene products. None of the mutations specifically inactivated the ade6-M26 hotspot. Additional rec genes may be identified with these methods.

Genetic and physical analysis of the M26 recombination hotspot of Schizosaccharomyces pombe.

The ade6-M26 mutation of Schizosaccharomyces pombe has previously been reported to stimulate ade6 intragenic meiotic recombination. We report here that the ade6-M26 mutation is a single G----T nucleotide change, that M26 stimulated recombination within ade6 but not at other distinct loci, and that M26 stimulated meiotic but not mitotic recombination. In addition, M26 stimulated recombination within ade6 when M26 is homozygous; this result demonstrates that a base-pair mismatch at the M26 site was not required for the stimulation. These results are consistent with the ade6-M26 mutation creating a meiotic recombination initiation site.

Chi-dependent DNA strand cleavage by RecBC enzyme.

Chi sites enhance in their vicinity homologous recombination by the E. coli RecBC pathway. We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E. coli recC gene, coding for a RecBC enzyme subunit. Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA. These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.

RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting.

Homologous recombination by the E. coli RecBC pathway occurs at elevated frequency near Chi sites. We reported previously that Chi induces RecBC enzyme to cleave one DNA strand--that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3' side of the Chi octamer and produces nicks with 3'-OH and 5'-PO4 groups. Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate. A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment. These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity. These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left. We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity.

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.

In vivo tyrosine phosphorylations of the Abelson virus transforming protein are absent in its normal cellular homolog.

The transforming gene product of the Abelson murine leukemia virus (A-MuLV) is a phosphoprotein encoded by combined viral and cellular sequences. Previous work has shown the existence of a serologically crossreactive normal cellular phosphoprotein called NCP150. We have utilized two-dimensional phosphopeptide mapping and phosphoamino acid analysis to compare the structures of NCP150 and wild-type and mutant forms of the A-MuLV protein labeled in vivo with 32P-orthophosphate. This analysis demonstrated clear homology between NCP150 and wild-type A-MuLV protein, but a number of phosphorylation differences were seen. Among them, two specific tyrosine phosphorylations present in all transformation-competent Abelson proteins were not observed in NCP150. No other phosphotyrosine-containing peptides were detected. In addition, transformation-defective mutants isolated from either the P120 or P160 wild-type strain lack phosphotyrosine-containing peptides. Double-infection studies with such transformation-defective and transformation-competent A-MuLV strains show that Abelson viral proteins may be substrates for their own tyrosine-specific kinase activity in vivo. These observations suggest that the phosphotyrosine kinase activity of the abl region may be controlled, and may function, differently in its viral and cellular forms.