Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations.To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.Many eukaryotic genomes contain families of moderately to highly repeated DNA elements containing RNA polymerase III (pol III) promoters (reviewed in references 74 and 75). Frequently these elements resemble the intragenic pol III promoter class found in tRNA and 7SL RNA genes, which consist of consensus A-box and B-box sequences downstream from the transcription start sites. These elements can be found either dispersed as individual copies or as highly reiterated tandem copies, especially in heterochromatic regions. The pol III elements are not generally transcribed into stable RNA commensurate with their copy number in vivo, although they can usually be transcribed in vitro, and there are numerous reports of condition-specific or development-specific activation in vivo (10,27,61,90,95,97,101). Several hypotheses have been put forward regarding possible functions for these sequences, but one particularly interesting suggestion is that dispersed RNA pol III promoters might exert either a positive or negative influence on the transcriptional activity of overlapping or nearby RNA pol II promoters (11,12,15,89,90,96). In some cases, cryptic pol III promoter elements directly interfere with factor binding sites in the pol II promoter upstream region or with the pol II initiation site itself. In at least one report, however, repression was achieved by an Alu repetitive element, in which case there was no obvious steric overlap with the neighboring pol II promoter (96).In this report, the question of whether RNA pol III promoters can exert negative transcriptional regulation on neighboring DNA has been approached by studying the budding yeast Saccharomyces cerevisiae. Although this yeast does not appear to have any Alu-ty...

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.nucleolus ͉ RNA polymerase III I t has previously been shown that tRNA-class RNA polymerase III (pol III) promoters can exert negative transcriptional regulation on neighboring DNA in the budding yeast, Saccharomyces cerevisiae (1). The degree to which this tRNA genemediated silencing (tgm silencing) affects nearby RNA polymerase II-transcribed genes varies among different pol II promoters. Although complete inhibition of some nearby pol II promoters has been achieved in selected artificial juxtapositions, promoters that are found bordering the 274 tRNA genes in their normal chromosomal locations must somehow be exempt, or at least conditionally resistant to this form of negative regulation.There is a particularly close association of tRNA genes with naturally occurring copies of most classes of the Ty retrotransposons (2). One possible selective pressure for maintaining an association between tRNA genes and neighboring pol II promoters is that the proximity serves some regulatory function that is beneficial for maintenance of the retrotransposon at that position. Although the presence of a Ty3 sigma element does not strongly affect expression of a neighboring tRNA gene (3), temperature-dependent silencing of the chromosomal Ty3 and sigma elements was shown to be dependent on RNA polymerase III, suggesting possible involvement of the neighboring tRNA transcription units (1). It is interesting to note that the one class of Ty retrotransposon that is not found adjacent to tRNA genes, the Ty5 class, is found instead at other silenced locations, namely telomeres and the silent mating type loci (4, 5).At this time, it is not clear what relationship the mechanism of tgm silencing might have to silencing at silent mating type loci, telomeres, ribosomal RNA genes, or other forms of negative regulation, but several types of interference between the transcription units appear to be ruled out. It seems unlikely that either readthrough by pol III or positive supercoils propaga...

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To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.The eukaryotic mRNA transcription apparatus comprises RNA polymerase II, general transcription factors and their associated factors, and gene-specific factors (reviewed in references 6, 13, and 23). RNA polymerase II (pol II) is a multisubunit enzyme with ϳ12 to 15 subunits, depending on the organism, that plays a major role in mRNA synthesis since it possesses the catalytic machinery for the formation of the 3Ј-5Ј phosphodiester bonds between ribonucleoside triphosphates and presumably responds to signals from the multiple factors involved in regulating its function during initiation and elongation of mRNA synthesis.Yeast Saccharomyces cerevisiae pol II has been a useful paradigm for enzyme function since its subunit structure and amino acid sequences are strikingly similar to pol II subunits from a variety of eukaryotes and even archaebacteria. S. cerevisiae pol II has 12 subunits, designated RPB1-RPB12, ranging in size from ϳ190 to ϳ8 kDa (reviewed in references 26 and 27). Five of these subunits (RPB5, RPB6, RPB8, RPB10, and RPB12) are also present in RNA polymerases I and III and are usually referred to as the common subunits.Since the functions of most pol II subunits are unclear, one approach to understanding their role in transcription has been to isolate the genes encoding human RNA polymerase subunits and test for functional similarities by determining if the human subunit can function in place of its yeast homolog. Only a few of the human subunit genes have been tested for heterocomplementation in S. cerevisiae. hsRPB6 has a highly conserve...

No abstract

Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated. In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo. This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites. Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations. To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements. This class of retrotransposons is always found integrated immediately upstream of different tRNA genes. Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold.