Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

Dieci, Giorgio; Giuliodori, Silvia; Catellani, Manuela; Percudani, Riccardo; Ottonello, Simone

doi:10.1074/jbc.m105036200

Cited by 44 publications

(51 citation statements)

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“…TFIIIC is not required for transcriptional reinitiation in vitro, and the presence of its binding sites (A and B boxes) within the transcribed region have led to the proposal that TFIIIC might be removed as the elongating polymerase transcribes through its binding site, leaving a Pol III-TFIIIB reinitiation complex (4,30,33,34). Our occupancy data provides in vivo support for this proposal, and is consistent with the following model (see Fig.…”

Section: Discussionsupporting

confidence: 76%

“…In addition to Pol III targets, several hundred Pol II targets were occupied by TBP above the cutoff value. Remarkably, Pol III targets are transcribed at levels of up to 50 RNAs per min (30), suggesting that high levels of transcription might require the constant presence of TBP. Consistent with this idea, the Pol II genes identified as highly occupied are highly transcribed (median of 50 mRNA transcripts per h) in comparison to all Pol II genes (median of 2 mRNAs per h), based on the activity estimates of others (29).…”

Section: Discussionmentioning

confidence: 99%

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The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

Roberts

Stewart

Huff

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

165

203

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RNA polymerase III (Pol III) transcribes small untranslated RNAs, such as tRNAs. To define the Pol III transcriptome in Saccharomyces cerevisiae, we performed genome-wide chromatin immunoprecipitation using subunits of Pol III, TFIIIB and TFIIIC. Virtually all of the predicted targets of Pol III, as well as several novel candidates, were occupied by Pol III machinery. Interestingly, TATA boxbinding protein occupancy was greater at Pol III targets than virtually all Pol II targets, and the highly occupied Pol II targets are generally strongly transcribed. The temporal relationships between factor occupancy and gene activity were then investigated at selected targets. Nutrient deprivation rapidly reduced both Pol III transcription and Pol III occupancy of both a tRNA gene and RPR1. In contrast, TFIIIB remained bound, suggesting that TFIIIB release is not a critical aspect of the onset of repression. Remarkably, TFIIIC occupancy increased dramatically during repression. Nutrient addition generally reestablished transcription and initial occupancy levels. Our results are consistent with active Pol III displacing TFIIIC, and with inactivation͞release of Pol III enabling TFIIIC to bind, marking targets for later activation. These studies reveal new aspects of the kinetics, dynamics, and targets of the Pol III system. R NA polymerase III (Pol III) in Saccharomyces cerevisiae transcribes genes for small structural RNAs important for many cellular processes. Among the known Pol III targets are structural RNAs for translation (all tRNAs, 5S ribosomal RNA, 7SL RNA), tRNA processing (RPR1; the RNA of the ribonuclease P complex), and splicing (i.e., U6). The S. cerevisiae genome is predicted to contain 280 targets, including 275 tRNA genes. The Pol III transcription machinery is highly conserved in eukaryotes, and consists of the multisubunit Pol III polymerase, two complexes (TFIIIB and TFIIIC) required for promoter recognition and͞or initiation, and an additional factor (TFIIIA) required only for 5S rDNA transcription (1-3).Several reviews have addressed the function and regulation of the Pol III machinery in detail (2-4), and here we provide a brief summary. Pol III promoters contain two conserved DNA sequence elements, termed A and B boxes, which are located within the transcribed region and direct the Pol III machinery to target genes (5, 6). The A and B boxes are recognized by the TFIIIC complex, which can bind in the absence of TFIIIB and Pol III, establishing TFIIIC as the promoter specificity factor (7-11). Because most Pol III targets are relatively short (Ͻ100 bases), TFIIIC can encompass nearly the entire gene (12).TFIIIC interacts with TFIIIB, enabling recruitment of TFIIIB to Pol III promoters (13,14). TFIIIB contains the subunits Brf1 (TFIIB-related factor), Bdp1͞Tfc5͞BЉ, and the TATA-binding protein (TBP). TBP is the only subunit of the basal factors not dedicated solely to Pol III transcription; TBP is used by all three RNA polymerases and is required for transcription at both TATAcontaining and TATA-less pr...

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

Roberts

Stewart

Huff

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

165

203

View full text Add to dashboard Cite

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“…All of the amplification products were inserted into pUC-derived plasmid vectors and sequence-verified by dideoxy chain termination sequencing. The SCR1⌬_T7 construct is the previously described 3Ј⌬ϩ90 variant of SCR1 (26). For in vivo analysis, this SCR1 gene variant was subcloned into the YEp352 shuttle vector.…”

Section: Methodsmentioning

confidence: 99%

“…To further test for possible effects of the upstream flanking sequence on T 5 -dependent termination, we generated a construct (SCR1⌬_3ЈL) in which the L(CAA)LR2 T 5 terminator, plus 20 bp of its natural 3Ј-flanking sequence followed by T 10 , were fused to the first 90 bp of the SCR1 gene, coding for the S. cerevisiae 7SL RNA. Apart from the presence of A-and B-blocks allowing for efficient in vitro transcription by the Pol III system (26), this portion of the SCR1 gene is essentially unrelated to tRNA coding sequences. As shown in Fig.…”

Section: Effect Of the Upstream Sequence Context On T 5 Terminatormentioning

confidence: 99%

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

Braglia

Percudani

Dieci

2005

Journal of Biological Chemistry

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104

112

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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 terminationproficient 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.Eukaryotic RNA polymerase (Pol) 1 III is unique among DNAdependent RNA polymerases in recognizing a simple run of T residues on the coding strand as a termination signal, in the apparent absence of accessory factors. The minimal signal thought to be sufficient to provoke Pol III termination varies among different eukaryotes. T 4 suffices for termination by Xenopus and human Pol III (1, 2), T 4 /T 5 for Schizosaccharomyces pombe Pol III (3), and T 5 /T 6 for Saccharomyces cerevisiae Pol III (4). Pol III termination within T clusters is generally heterogeneous and progressive (5), and termination efficiency tends to increase with the length of the T run (4). Mechanistically, Pol III termination involves extensive pausing, dictated by the T cluster itself (5, 6). Even during elongation, the addition of three UMP residues in succession is particularly slow (5). Pol III pausing at T clusters sets cycles of hydrolytic RNA chain retraction (7). Both pausing and the associated hydrolytic RNA cleavage are affected by mutations in the C160, C128, and C11 subunits of yeast Pol III (7-9). In the case of C128 and C11, the same mutations have also been shown to affect the termination properties of Pol III, thus suggesting a functional interplay between hydrolytic RNA cleavage and transcription termination (9, 10). The t...

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“…In some of these genes, the TFIIIC-interacting control regions (box A and box B) have been maintained within the transcribed region, and adapted to the structure of the small RNA without losing the transcriptional function. For example, in the Saccharomyces cerevisiae SCR1 gene, coding for the 7SL RNA, box A and box B are both intragenic and involved in transcription, yet their sequences display distinguishing features as compared with the consensus of the tDNA box A and box B (2). In the S. cerevisiae SNR6 gene, coding for the U6 snRNA, a non-canonical, yet transcriptionally relevant box A has been maintained within the transcribed region, whereas the box B is located downstream of the transcription termination site (3,4).…”

mentioning

confidence: 99%

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

Guffanti

Ferrari

Preti

et al. 2006

Journal of Biological Chemistry

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The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA Asn (GTT) gene family, but not for two tRNA Pro (AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA Asn genes, but only in the presence of TFIIIB reconstituted with a crude B؆ fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA Asn genes was strongly influenced by their 5-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIICinteracting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.RNA polymerase (Pol) 3 III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs. In general, the transcription of class III genes is under the control of internal control regions (ICR) characterized by discontinuous structures, with essential boxes separated by non-essential nucleotides (1). In the case of tRNA and 5 S rRNA genes, the ICRs are highly conserved and comprise the binding sites for the general transcription factor TFIIIC (box A and box B) and for the 5 S-specific factor TFIIIA (box C). Once assembled, TFIIIC recruits TFIIIB upstream of the transcription start site (TSS); TFIIIB in turn recruits Pol III for transcription initiation. The strong conservation of the ICRs likely reflects their dual function as both nucleation sites for transcription complex assembly and key determinants of tRNA and 5 S rRNA structure. An indication of the constraints imposed on ICR by their overlapping structural and functional roles comes from the variability of promoter organizations displayed by the minority of class III genes not coding for tRNA and 5 S rRNA. In some of these genes, the TFIIIC-interacting control regions (box A and box B) have been maintained within the transcribed region, and adapted to the structure of the small RNA without losing the transcriptional function. For example, in the Saccharomyces cerevisiae SCR1 gene, coding for the 7SL RNA, box A and box B are both intragenic an...

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Intragenic Promoter Adaptation and Facilitated RNA Polymerase III Recycling in the Transcription of SCR1, the 7SL RNA Gene ofSaccharomyces cerevisiae

Cited by 44 publications

References 50 publications

The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships

Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III

A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

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