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At least 19 recordsLinked to original sources

PubMed2006

Spatial organization of transcription by RNA polymerase III.

RNA polymerase III (pol III) transcribes many essential, small, noncoding RNAs, including the 5S rRNAs and tRNAs. While most pol III-transcribed genes are found scattered throughout the linear chromosome maps or in multiple linear clusters, there is increasing evidence that many of these genes prefer to be spatially clustered, often at or near the nucleolus. This association could create an environment that fosters the coregulation of transcription by pol III with transcription of the large ribosomal RNA repeats by RNA polymerase I (pol I) within the nucleolus. Given the high number of pol III-transcribed genes in all eukaryotic genomes, the spatial organization of these genes is likely to affect a large portion of the other genes in a genome. In this Survey and Summary we analyze the reports regarding the spatial organization of pol III genes and address the potential influence of this organization on transcriptional regulation.

Animals↗

PubMed1999

Termination sequence requirements vary among genes transcribed by RNA polymerase III.

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.

Animals↗

PubMed2006

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III.

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.

Animals↗

PubMed2001

Superimposed promoter sequences of the adenoviral E2 early RNA polymerase III and RNA polymerase II transcription units.

The human adenovirus type 2 E2 early (E2E) transcriptional control region contains an efficient RNA polymerase III promoter, in addition to the well characterized promoter for RNA polymerase II. To determine whether this promoter includes intragenic sequences, we examined the effects of precise substitutions introduced between positions +2 and +62 on E2E transcription in an RNA polymerase III-specific, in vitro system. Two noncontiguous sequences within this region were necessary for efficient or accurate transcription by this enzyme. The sequence and properties of the functional element proximal to the sites of initiation identified it as an A box. Although a B box sequence could not be unambiguously located, substitutions between positions +42 and +62 that severely impaired transcription also inhibited binding of the human general initiation protein TFIIIC. Thus, this region of the RNA polymerase III E2E promoter contains a B box sequence. We also identified previously unrecognized intragenic sequences of the E2E RNA polymerase II promoter. In conjunction with our previous observations, these data establish that RNA polymerase II and RNA polymerase III promoter sequences are superimposed from approximately positions -30 to +20 of the complex E2E transcriptional control region. The alterations in transcription induced by certain mutations suggest that components of the RNA polymerase II and RNA polymerase III transcriptional machines compete for access to overlapping binding sites in the E2E template.

Adenovirus E2 Proteins↗

PubMed1995

Both RNA polymerase III and RNA polymerase II accurately initiate transcription from a human U6 promoter in vitro.

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box. Both pol III- and pol II-specific transcription were stimulated by addition of recombinant TATA-binding protein (TBP). We conclude that both pol III and pol II preinitiation complexes can assemble on the U6 promoter in vitro and could compete during the bona fide process in the cell.

Animals↗

PubMed2001

RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. Several DNA elements have been identified that act to separate these chromatin domains. We report a detailed characterization of one of these elements, identifying it as a unique tRNA gene possessing the ability to block the spread of silent chromatin in Saccharomyces cerevisiae efficiently. Transcriptional potential of the tRNA gene is critical for barrier activity, as mutations in the tRNA promoter elements, or in extragenic loci that inhibit RNA polymerase III complex assembly, reduce barrier activity. Also, we have reconstituted the Drosophila gypsy element as a heterochromatin barrier in yeast, and have identified other yeast sequences, including the CHA1 upstream activating sequence, that function as barrier elements. Extragenic mutations in the acetyltransferase genes SAS2 and GCN5 also reduce tRNA barrier activity, and tethering of a GAL4/SAS2 fusion creates a robust barrier. We propose that silencing mediated by the Sir proteins competes with barrier element-associated chromatin remodeling activity.

Acetyltransferases↗

PubMed2002

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits.

Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.

Amino Acid Sequence↗

PubMed1995

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.

Base Sequence↗

PubMed1984

Construction and functional analysis of a series of synthetic RNA polymerase III promoters.

RNA polymerase III promoters were constructed by cloning chemically synthesized double stranded analogues of the box A and box B consensus sequences into suitable vectors. In contrast to approaches adopted previously for the analysis of RNA polymerase III promoters, this method has no limitation on the structure and number of variants generated, and allows critical sequences in various permutations to be studied. Furthermore, the series of synthetic polymerase III promoters created constitute a collection of point mutation variants and hence provide a powerful tool for the analysis of nucleotides essential for promoter function. The results demonstrate that these two boxes, when separated by approximately 50 base pairs, are sufficient to direct efficient transcription, and that substitution of certain nucleotides causes reduced template activity.

Base Sequence↗

PubMed1984

Construction and functional analysis of a series of synthetic RNA polymerase III promoters.

RNA polymerase III promoters were constructed by cloning chemically synthesized double-stranded analogues of the box A and box B consensus sequences into suitable vectors. In contrast to approaches adopted previously for the analysis of RNA polymerase III promoters, this method has no limitation on the structure and number of variants generated and allows critical sequences in various permutations to be studied. Furthermore, the series of synthetic polymerase III promoters created constitute a collection of point mutation variants and hence provide a powerful tool for the analysis of nucleotides essential for promoter function. The results demonstrate that these two boxes, when separated by 51 base pairs, are sufficient to direct efficient transcription and that substitution of certain nucleotides causes reduced template activity.

Base Sequence↗

PubMed2005

Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III.

Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T(5) and T(4) are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T(5) are very rare in mammals. In Saccharomyces cerevisiae, the strength of T(5) as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T(5) in the 3'-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T(5). In contrast, tDNA transcription termination was highly efficient when T(5) was followed by an A or G residue. Surprisingly, however, when a termination-proficient T(5) signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T(6) sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.

Animals↗

PubMed2006

Transcription factor TFIIIB and transcription by RNA polymerase III.

pol (RNA polymerase) III is charged with the task of transcribing nuclear genes encoding diverse small structural and catalytic RNAs. We present a brief review of the current understanding of several aspects of the pol III transcription apparatus. The focus is on yeast and, more specifically, on Saccharomyces cerevisiae; preponderant attention is given to the TFs (transcription initiation factors) and especially to TFIIIB, which is the core pol III initiation factor by virtue of its role in recruiting pol III to the transcriptional start site and its essential roles in forming the transcription-ready open promoter complex. Certain relatively recent developments are also selected for brief comment: (i) the genome-wide analysis of occupancy of pol III-transcribed genes (and other loci) by the transcription apparatus and the location of pol III transcription in the cell; (ii) progress toward a mechanistic and molecular understanding of the regulation of transcription by pol III in yeast; and (iii) recent experiments identifying a high mobility group protein as a fidelity factor that assures selection of the precise transcriptional start site at certain pol III promoters.

Gene Expression Regulation↗

PubMed2000

Identification of a negative regulatory DNA element for neuronal BC1 RNA expression by RNA polymerase III.

BC1 RNA is a neuronal cell-specific RNA polymerase III (Pol III) transcript. The BC1 RNA gene has plural types of Pol III promoters, in addition to which an E-box sequence (E2 site) acts as a transcriptional activator, which is recognized by a brain-specific protein(s). Using an in vitro transcription system, we found that the upstream region of the BC1 RNA gene contained a sequence that interfered with the activity of the E-box element in a distance-independent manner. A tandem repeat within this sequence, which was weakly homologous with the neuron-restrictive silencer element (NRSE) found in the Pol II system, was recognized by a brain nuclear protein. Consistently, the transcriptional activity increased by deleting the tandem repeat sequence. We called this BC1 RNA-repressing element BCRE. The DNA-binding specificities of BCRE-binding protein differed from that of NRSE-binding protein (NRSF). A similar protein with an ability to bind to BCRE was also found in liver and kidney. Furthermore, the glutamate analog kainic acid increased the DNA-binding of both E2 site-binding protein and BCRE-binding protein, and then the levels of BC1 RNA also increased transiently. Our results suggested that both positive and negative regulatory elements contribute to neuronal BC1 RNA expression.

Animals↗

PubMed1987

The capped U6 small nuclear RNA is transcribed by RNA polymerase III.

U6 RNA is an abundant, capped, small nuclear RNA (snRNA) species associated with heterogeneous nuclear ribonucleoproteins in eukaryotic cells. U4 RNA and U6 RNA are hydrogen bonded in a 1:1 ratio in discrete small nuclear ribonucleoprotein particles that are required in pre-mRNA processing. Previous reports have established that the mRNAs and U1 to U5 U-snRNAs are synthesized by RNA polymerase II. Evidence is presented here for synthesis of U6 RNA by RNA polymerase III. The synthesis of U6 RNA in vitro, using Novikoff hepatoma or HeLa whole cell extracts, was not inhibited at low (1 microgram/ml) concentrations of alpha-amanitin, and only 35% inhibition occurred at 10 micrograms/ml concentration. The in vitro synthesized U6 RNA, like other RNA polymerase III transcripts, was associated with La antigen. The U6 RNA synthesized in vitro by the whole cell extracts was capped, but no other internal post-transcriptional modifications were found. Uridylic acid residues were also added post-transcriptionally to the 3'-end of U6 RNA in vitro. U6 RNA, though capped on its 5'-end, is transcribed by RNA polymerase III; this is the first report of a capped RNA molecule synthesized by RNA polymerase III.

Amanitins↗

PubMed1978

Structural relationships of low molecular weight viral RNAs synthesized by RNA polymerase III in nuclei from adenovirus 2-infected cells.

Previous studies have shown that endogenous class III RNA polymerase(s) in nuclei from adenovirus 2-infected cells synthesize virus-coded RNA species which are approximately 200 (V200), 156 (V156), and 140 (V140) nucleotides in length (Weinmann, R., Brendler, T. G., Raskas, H.J., and Roeder, R. G. (1976) Cell 7, 557-566). The V156 nuclear RNA is identical in sequence to the major virus-associated RNA (VA RNA1 or 5.5 S RNA) synthesized in intact cells (Ohe, K., and Weissman, S. M. (1971) J. Biol. Chem. 246, 6991-7009). The V140 RNA contains several components, one of which appears identical to a minor virus-associated RNA (VA RNAII) which is synthesized in infected cells (Mathews), M. B. (1975) Cell 6, 223-229). Thus transcription of the VA RNAI and VA RNAII genes in vitro accurately reflects the in vivo transcription of these genes. The V200 RNA contains all the nucleotide sequences found in V156 RNA plus an additional 38 to 40 nucleotides on the 3' terminus. Transcription of the gene encoding this RNA species terminates within a stretch of 6 deoxythymidylic acid residues which are located 38 nucleotides beyond the predicted termination site for VA RNAI and which are preceded by a GC-rich sequence of nucleotides. These data suggest either that the V200 RNA is a precursor to the VA RNAI or that the RNA polymerase III occasionally reads-through the presumptive VA RNAI gene termination signal and stops at a potentially stronger downstream termination site.

Adenoviridae↗

PubMed1997

Substrate specificity of the RNase activity of yeast RNA polymerase III.

Using yeast RNA polymerase III ternary complexes stalled at various positions on the template, we have analyzed the cleavage products that are retained and released by the transcription complexes. The retained 5' products result from cleavage at uridine residues during retraction, whereas the yield of mononucleotides and dinucleotides released indicates that multiple cuts occur near the 3' end. Comparison of the cleavage patterns of uridine-containing and 5-bromouridine-containing transcripts suggests that RNA within an RNA-DNA hybrid duplex is the substrate for the 3'-5' exonuclease. During transcription of the SUP4 tRNATyr gene, RNA polymerase III produces not only full-length pre-tRNATyr but also short oligonucleotides, indicating that exonuclease digestion and transcription are concurrent processes. To explore the possibility that these oligonucleotides are released by the action of the RNA polymerase III nuclease at previously observed uridine-rich pause sites, we tested modified templates lacking the arrest sites present in the SUP4 tRNATyr gene. Comparative studies of cleavage during transcription for these templates show a direct correlation between the number of natural pause sites and the yield of 3' products made. At the natural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short oligoribonucleotides with uridine residues at the 3' terminus.

Base Sequence↗

PubMed1994

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7.

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

Amino Acid Sequence↗

PubMed2006

Coordinated nuclear import of RNA polymerase III subunits.

Eukaryotic RNA polymerases are multisubunit assemblies, whose enzymatic function in the nucleus is intensively studied. However, little is known about the biogenesis of the three RNA polymerases and coupling to nucleo-cytoplasmic transport. Here, we show that Rpc128, the second largest subunit of RNA polymerase III, was mislocalized to the cytoplasm, when a short sequence in the N-terminal domain was deleted. Importantly, nuclear import of other, but not all, RNA polymerase III subunits was impaired in this RPC128DeltaN mutant. These data suggest that RNA polymerase III subunits are not imported independently into the nucleus but may require preassembly into cytoplasmic subcomplexes for coordinated nuclear uptake. We expect these studies to be a starting point to dissect the complex biogenesis pathway of eukaryotic RNA polymerases.

Active Transport, Cell Nucleus↗