Formation Of Stable Transcription Complexes Research Articles

UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat.

The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.

The Roles of Specific Template Nucleosides in the Formation of Stable Transcription Complexes by Escherichia coli RNA Polymerase

We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro. The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed. On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation. The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process. On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes. On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.

Ultrastructural allelic variation in HLA-DQB1 promoter elements

Sequence variation among HLA class II promoter elements may contribute to functional differences in transcriptional regulation of different class II alleles. In addition to influencing the binding sites for nuclear transcription factors, promoter polymorphism may also alter intrinsic structural properties of the DNA strands, such as conformation and curvature, which influence the formation of stable transcription complexes. We used SSCP analysis of PCR-amplified promoter regions from the DQB1 locus to evaluate conformational polymorphism within DQ alleles. Distinct electrophoretic migration patterns of the SSCP products were detected for six DQB1 alleles; analysis of the DQB 1 ∗0302 promoter, known to be associated with type 1 diabetes, showed no SSCP differences between IDDM patients and normal controls. Using computer modeling based on a “nearest-neighbor” energy of predicted curvature theory, we examined the effect of allelic promoter region sequence polymorphism on the predicted curvature of doublestranded DNA, and found distinct allelic differences in predicted DNA curvature, both in transcriptional consensus binding sites and in regions located between binding sites. These data are consistent with a model in which intrinsic sequence variation in the promoter region results in ultrastructural differences which may influence DNA bending and interactions with multimeric DNA-protein transcription complexes.

Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties.

RNA polymerase III transcription factor TFIIIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of 90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFIIIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90- and 70-kDa TFIIIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIIIC and DNA, stabilizes TFIIIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIIIB. The 90-kDa component of TFIIIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIIB and bestows heparin-insensitivity on the TFIIIB-DNA complex. The resolution of TFIIIB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.

The characterization of the TFIIIA synthesized in somatic cells of Xenopus laevis.

In somatic cells of Xenopus, transcription of the TFIIIA gene initiates greater than 200 bp upstream from the start site used in oocytes. The resultant mRNA encodes a protein, S-TFIIIA, that is 22 amino acids longer at its amino terminus than the abundant form of TFIIIA in oocytes (O-TFIIIA). S-TFIIIA binds the 5S RNA gene and 5S RNA, and both O- and S-TFIIIA promote the formation of stable transcription complexes on oocyte-type 5S RNA genes in an oocyte nuclear extract. We have not found any functional difference between the two forms of TFIIIA. Different transcription start sites suggest differential promoter usage--one in oocytes that permits high levels of gene activity and another that is used in somatic cells for low-level TFIIIA mRNA synthesis.

Reaction parameters of TFIIIA-induced supercoiling catalyzed by aXenopus Iaeviscell-free extract

In addition to its fundamental role of nucleating the formation of stable transcription complexes, the Xenopus laevis 5S RNA specific transcription factor, TFIIIA, promotes a variety of DNA-associated metabolic reactions. We report that TFIIIA can induce a DNA supercoiling catalyzed by the Xenopus laevis S-150 cell-free extract on plasmids containing a single copy of the Xenopus 5S RNA gene (somatic-type). Stimulated supercoiling occurs in the presence of high concentrations of ATP (4 mM) and at a factor to DNA ratio of 1 through a mechanism most likely involving type I topoisomerase. The highest level of stimulated supercoiling occurs when TFIIIA is incubated with DNA prior to the addition of the S-150 extract. Taken together, the experiments outlined in this report establish a reliable and seminal system in which TFIIIA-induced DNA supercoiling can be observed reproducibly.

Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.

Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.

Formation of stable transcription complexes as assayed by analysis of individual templates.

Conditions were established where transient transfection of two marker genes resulted in the expression of one or the other, but not both, in individual cells as assayed by immunofluorescence. Thus, the expression from a single cell reflects the activity of single active transcription templates. Under these conditions, a vector encoding the simian virus 40 large tumor antigen (SV40 T-Ag) driven by the SV40 enhancer and early promoter was transfected into CV-1, L, or HeLa cells yielding, for all three cell types, about 10-30% T-Ag-positive cells as assayed by immunofluorescence. Similar vectors containing either mutated or deleted SV40 enhancers also gave T-Ag-positive cells, but at about 1/100 the frequency. Quantitative analysis showed that T-Ag-positive cells produced about the same amount of T-Ag whether or not an active enhancer was present. Chloramphenicol acetyltransferase-encoding vectors gave the same result. The data are consistent with the hypothesis that at a low, but finite, probability, fully functional transcription complexes can form on a given active template in the absence of enhancer DNA. Enhancers seem to increase the number of active templates. Subcloning experiments suggest that these transcription complexes can be surprisingly stable.

Novobiocin inhibits interactions required for yeast TFIIIB sequestration during stable transcription complex formation in vitro.

Novobiocin concentrations normally used to inhibit a putative eukaryotic DNA gyrase have been found to inhibit transcription of a yeast 5S rRNA gene using an in vitro yeast transcription system. Purified RNA polymerase III and three yeast transcription factors (chromatographically separated, partially purified and free of any detectable gyrase activity) were used. Novobiocin prevents specific transcription if added to the in vitro system immediately prior to the addition of transcription factors and RNA polymerase. If a stable transcription factor complex is allowed to form prior to the addition of novobiocin, concentrations of novobiocin as high as 1000 micrograms/ml have no effect on in vitro transcription. Transcription factors TFIIIA and TFIIIC are able to be stably sequestered onto 5SrDNA-cellulose, but factor TFIIIB is not able to associate with the 5SrDNA-TFIIIA-TFIIIC complex in the presence of novobiocin. Although novobiocin is able to precipitate other basic proteins, it does not appear to precipitate any of these class III gene transcription factors, but instead appears to act by disrupting specific factor-factor interactions.

Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro.

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.

Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro.

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.

An enhancer-like sequence within the Xenopus U2 gene promoter facilitates the formation of stable transcription complexes

Enhancers are eukaryotic promoter elements that increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene. There is growing evidence that enhancer action is mediated by transacting factors, but the mode of action of these factors is not yet known. We report here on the Xenopus U2 gene promoter, which contains two sequence elements. The distal sequence element increases promoter activity 20-fold by facilitating the formation of stable transcription complexes. A synthetic 14-base-pair (bp) oligonucleotide corresponding to part of the distal sequence element, which shows homology to an immunoglobulin gene promoter element and to both the simian virus 40 (SV40) and the immunoglobulin heavy-chain gene enhancers, stimulates transcription in an orientation-independent manner.

Point mutations in the 5' ICR and anticodon region of a Drosophila tRNAArg gene decrease in vitro transcription.

We have examined the effects of various nucleotide substitutions in a Drosophila tRNAArg gene on in vitro transcription and stable transcription complex formation in Drosophila KcO and HeLa cell extracts. Substitutions in positions encoding the invariant G18 and G19 residues resulted in decreased transcription, however, the moderate decreases indicate that these nucleotides are not obligatory promoter recognition sites. An A21 to C21 mutation had no effect on transcription levels using homologous extract however, this mutant displayed decreased transcriptional abilities in HeLa cell extract. Nucleotide substitutions within the sequence encoding the anticodon led to a decrease in the transcription activity but not in the ability to form a stable transcription complex.

Characterisation of a Dictyostelium discoideum DNA fragment coding for a putative tRNAValGUU gene.

A genomic DNA fragment from Dictyostelium discoideum was characterized. This DNA, although 74% d(A + T)-rich, codes for a putative tRNAValGUU. The tRNAVal gene overlaps at its 5' half with another RNA polymerase III transcription unit. This RNA polymerase III transcription unit can be folded into a tRNA-like shape and is comprised of significant amounts of invariant and semi-invariant nucleotides present in all eukaryotic tRNAs. This unit contains the two promoter blocks defined for RNA polymerase III, which are homologous to recently defined promoter elements to the extent of 76-88% (A block) and 86-93% (B block) respectively [Sharp et al. (1981) Proc. Natl Acad. Sci. USA 78, 6657-6661]. Both of the overlapping class III genes are transcribed in germinal vesicle extracts prepared from Xenopus laevis oocytes as a single transcription unit, resulting in an unusually large product compared to primary transcripts of other tRNA genes. The unit is not transcribed in HeLa extracts but it competes very strongly for transcription factor(s) under the conditions of stable transcription complex formation. Although the whole unit is transcribed, it is believed that only one functional product is formed. Therefore we define the tRNA-like structure, coded for on this class III transcription unit, as a putative tRNA 'pseudogene' meaning that, although it is transcribed by RNA polymerase III, it is not likely to mature to a functional tRNA.

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene.

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene.

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

HeLa cell RNA polymerase III transcription factors. Functional characterization of a fraction identified by its activity in a second template rescue assay.

That stable transcription complexes are formed in HeLa cell RNA polymerase III transcription extracts (Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173) can be shown in order of template addition experiments with DNAs coding for adenovirus 2 VA I RNA or one of the Bombyx mori tRNAAla2 genes. Using this property of the HeLa extracts in a "second template rescue assay" has allowed us to partially purify protein components involved in stable transcription complex formation. Two fractions, called transcription fraction X (TfrX) and transcription fraction Y (TfrY), are required to fully reconstitute selective transcription of the VA I and tRNAAla2 DNA templates. TfrX contains one or more components required for forming transcriptional pre-emptive complexes, as shown in order of addition experiments. TfrX strongly protects a DNA segment surrounding the highly conserved distal sequence (the so-called B block) of the VA I and tRNAAla2 genes from DNase I digestion; we have also characterized weak protection of other segments of these genes by TfrX. DNase I protection experiments with TfrX and probes prepared from deletion variants of the VA I gene show that an intact B block is required for the strong protection.

Competition between Xenopus satellite I sequences and Pol III genes for stable transcription complex formation.

We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans. Transcription of a tRNA gene is also precluded by satellite I DNA. The Xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the satellite promoter is largely temperature independent over the range of 0-20 degrees, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants.

Partial purification of Drosophila Kc cell RNA polymerase III transcription components. Evidence for shared 5 S RNA and tRNA gene factors.

Class III transcription components were fractionated from Drosophila Kc cells. By the use of successive DEAE-Sephadex and CM-Sepharose column chromatographies, two fractions were identified to contain distinct positive transcription components. The reconstitution of these two fractions and RNA polymerase III was essential for faithful in vitro transcription of cloned Drosophila tRNA and 5 S RNA genes. Further chromatography of one of these fractions using phosphocellulose and DEAE-cellulose did not separate additional components required for reconstitution of transcription. These fractions may contain generalized class III transcription factors, as suggested by the cofractionation of required tRNA and 5 S RNA transcription components. We present further evidence that tRNA and 5 S RNA genes share at least one common transcription component other than RNA polymerase III by the use of stable transcription complex formation and competition experiments.