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Query: UNIPROT:P06889 (Mol)
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We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.
J Mol Biol 1992 Nov 20
PMID:A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III. 145 50

Locations of component proteins of yeast RNA polymerase III transcription factors (TFIII) A, C and B on a 5 S rRNA gene have been determined by site-specific DNA-protein photo-crosslinking. Comparison with a previously analyzed tRNA gene shows that similar nucleoprotein structures assemble on these two genes despite their differently located internal promoter elements. A principal signature of this homology is the placement of the 95 kDA subunit of TFIIIC, which associates with the box A promoter element of the tRNA gene. On the 5 S rRNA gene, the 95 kDa subunit occupies the same space in the absence of a box A sequence, and despite the presence of a box A-like sequence 30 base-pairs further downstream. A 90 kDa component that was not previously recognized as an integral part of TFIIIC has been specifically located at the 3' end of the 5 S rRNA gene.
J Mol Biol 1992 Dec 20
PMID:Topography of transcription factor complexes on the Saccharomyces cerevisiae 5 S RNA gene. 147 78

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.
Mol Cell Biol 1992 Sep
PMID:A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo. 150 99

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.
Plant Mol Biol 1992 Sep
PMID:Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III. 151 Nov 42

The promoters of both RNA polymerase II- and RNA polymerase III-transcribed small nuclear RNA (snRNA) genes contain an essential and highly conserved proximal sequence element (PSE) approximately 55 bp upstream from the transcription start site. In addition, the upstream enhancers of all snRNA genes contain binding sites for octamer-binding transcription factors (Octs), and functional studies have indicated that the PSE and octamer elements work cooperatively. The present study has identified and characterized a novel transcription factor (designated PTF) which specifically binds to the PSE sequence of both RNA polymerase II- and RNA polymerase III-transcribed snRNA genes. PTF binding is markedly potentiated by Oct binding to an adjacent octamer site. This potentiation is effected by Oct-1, Oct-2, or the conserved POU domain of these factors. In agreement with these results and despite the independent binding of Octs to the promoter, PTF and Oct-1 enhance transcription from the 7SK promoter in an interdependent manner. Moreover, the POU domain of Oct-1 is sufficient for significant in vitro activity in the presence of PTF. These results suggest that essential activation domains reside in PTF and that the potentiation of PTF binding by Octs plays a key role in the function of octamer-containing snRNA gene enhancers.
Mol Cell Biol 1992 Jul
PMID:Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes. 153 87

The dispersion of short interspersed elements (SINEs) probably occurred through an RNA intermediate. B1 is a murine homolog of the human SINE Alu; these elements are composed of 5' G + C-rich regions juxtaposed to A-rich tracts and are flanked by direct repeats. Internal promoters direct RNA polymerase III to transcribe B1 and Alu elements and proceed into the 3' flanking DNA until it reaches a (dT)4 termination signal. The resulting transcripts contain 3'-terminal oligo(U) tracts which can presumably base pair with the A-rich tract to form self-primed templates for reverse transcriptase and retrotransposition. Nuclear extracts from mouse tissue culture cells contain an RNA processing activity that removes the A-rich and 3'-terminal regions from purified B1 RNAs (R. Maraia, Nucleic Acids Res. 19:5695-5702, 1991). In this study, we examined transcription and RNA processing in these nuclear extracts. In contrast to results with use of purified RNA, nascent transcripts synthesized in nuclear extract by RNA polymerase III are not processed, suggesting that the transposition-intermediate-like RNA is shielded from processing by a protein(s). Alteration of an AATTTT TAA termination signal to a GCTTTTGC signal activated processing by greater than 100-fold in coupled transcription/processing reactions. A similar difference was found when expression was compared in frog oocytes. No difference in processing was found if the transcripts were made by T7 RNA polymerase in the presence of the nuclear extract, indicating that the different processing effects of the two terminators were dependent on synthesis by polymerase III. The modulation of processing of B1-Alu transcripts and the potential for retrotransposition of B1 and Alu DNA sequences are discussed.
Mol Cell Biol 1992 Apr
PMID:The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product. 154 7

Transcription elongation catalysed by DNA-dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid-encoded ribosomal RNA operon. Plasmid templates with different superhelical densities (sigma between +0.0017 and -0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.
Mol Microbiol 1992 Mar
PMID:Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region. 155 58

We have recently identified a tRNA gene cluster in the Arabidopsis nuclear genome. One tRNA(Ser) (AGA) gene and two tRNA(Tyr) (GTA) genes occur in tandem arrangement on a 1.5 kb unit that is amplified about 20-fold at a single chromosomal site. Here we have studied the in vitro expression of seven individually cloned tRNA(Ser) genes (pAtS1 to pAtS7) derived from this cluster. Five out of the seven tRNA(Ser) genes contain point mutations in the coding region which have in part adverse effects on the expression of these genes in different cell-free systems: (i) C10 and A62 in plant tRNA(Ser) genes, which correspond to G10 and C62, respectively, in all known vertebrate tRNA genes, result in a reduced transcription efficiency in HeLa but not in yeast extract. This indicates that yeast RNA polymerase III tolerates nucleotide substitutions at positions 10 [5' internal control region (ICR)] and 62 (3' ICR), whereas the vertebrate RNA polymerase III requires a more stringent consensus sequence. (ii) Processing of a pre-tRNA(Ser) with a mismatch in the aminoacyl stem is impaired in HeLa, yeast and wheat germ extracts, however, a mismatch in the anticodon stem is deleterious for HeLa and wheat germ but not for yeast processing enzymes. The unexpectedly high number of potential tRNA(Ser) pseudogenes in the cluster - quite in contrast to the tRNA(Ser) genes which mainly code for functional tRNAs - suggested that tRNA(Ser) (AGA) genes also occur elsewhere in the genome. We present evidence that single copies of tRNA(Ser) (AGA) genes do indeed exist outside the tRNA gene cluster.
Mol Gen Genet 1992 May
PMID:Expression of variant nuclear Arabidopsis tRNA(Ser) genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts. 160 62

The Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNA(Tyr) gene at 0 degrees C. RNA polymerase III specifically associates at 0 degrees C with this TFIIIC-TFIIIB-DNA complex to form a stable "closed" promoter complex in which the DNA surrounding the transcriptional start retains its duplex form. Promoter "opening" is a temperature-dependent and readily reversible process that involves up to 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO4 oxidation. This promoter opening increases progressively from 10 degrees C to 40 degrees C, with at least two regions within the transcription bubble appearing to melt independently. In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10 degrees C and 15 degrees C. When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides, there is no displacement of the transcription bubble. These transcription complexes are distinguishable from "open" promoter complexes in their maintenance of the transcription bubble at 0 degrees C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin. In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the S. cerevisiae-derived system. We show that if an RNA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RNase A, or RNase T1.
J Mol Biol 1992 Jul 05
PMID:Formation of open and elongating transcription complexes by RNA polymerase III. 161 62

The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.
Mol Gen Genet 1992 Jun
PMID:Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae. 162 93


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