The RNA polymerase III transcriptome revealed by genome-wide localization and activity-occupancy relationships - PubMed (original) (raw)
The RNA polymerase III transcriptome revealed by genome-wide localization and activity-occupancy relationships
Douglas N Roberts et al. Proc Natl Acad Sci U S A. 2003.
Abstract
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 box-binding 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 inactivation/release 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.
Figures
Fig. 1.
ChIP of the Pol III machinery enriches segments linked to Pol III genes. Spot/segment color reflects the relative enrichment of DNA homologous to the segment on the array (orange/red = high enrichment, see Results). An identical array grid of 30 spots/segments derived from each ChIP microarray experiment. Rows that contain a tRNA-linked segment are identified at right, along with the respective column number.
Fig. 2.
Alignment of ChIP microarray results with the physical map, centered on a representative tRNA target. A 10-kb segment of chromosome X is depicted. For each member of the Pol III machinery, spots/segments were culled from different locations on the same array and arranged on the grid according to the physical map.
Fig. 3.
Defining Pol III targets by percentile rank analysis. Percentile rank values for each segment were determined as described in Results. Average percentile rank values were plotted versus the number of segments with the same average percentile rank (frequency). (A) Segments from the untagged strain generate a Gaussian distribution curve. (B_–_G) Segments from tagged strains or using TBP antisera generate bimodal distribution curves. Those segments to the right of the trough (cutoff) are colored red and considered enriched. Cutoffs: Rpc82, 83%; Rpc40, 83%; Brf1, 82%; TBP, 85%; Tfc1, 83%; Tfc6, 81%.
Fig. 4.
Activity–occupancy analysis of the Pol III machinery at tRNAPhe(GAA)P2 in response to nutrient availability. (A) Relative transcript levels of tRNAPhe(GAA)P2 determined by Northern analysis of total RNA from strain YBC1846 (similar results were obtained with untagged wild-type strain FT4; data not shown) using a probe to the mature form of the tRNA to detect both the unspliced and spliced forms. A probe to Pol II-transcribed U4 serves as a control. (B) Multiplex PCR analysis of tRNAPhe(GAA)P2 occupancy (see supporting information for details). Titration of the input DNA establishes that amplification is linear in the range tested, and ChIP from an untagged strain (FT4) provides little or no product (lane 4). The asterisk in lane 4 indicates no antisera control. (C) The initial occupancy levels of members of the Pol III machinery in rich medium (T = 0 in the regimen). The amplicon for tRNAPhe(GAA)P2 encompasses the entire tRNA gene. The control amplicon for all subunits except TBP is within the Pol II-transcribed CDC2 ORF. For TBP, the control amplicon is within the TRA1 ORF to ensure that the distance to a TATA box is >4 kb. PCR product accumulation was quantified by Sybr Green fluorescence (see Methods). Error bars represent ±1 SD for triplicate qPCR quantitations of a representative experiment. These initial occupancy levels (T = 0) are set to 100% for D and E.(D and E) Occupancy of tRNAPhe(GAA)P2 and analysis of the corresponding transcript in response to nutrient availability. One member of each complex is shown in each graph for clarity. Data from a representative ChIP experiment of each subunit is provided. Error bars represent ± 1SD for triplicate qPCR quantitations of a representative experiment. Average quantitation (quantified from blot in A and a replicate, not shown) of the level of unspliced tRNAPhe(GAA)P2 versus U4 from two independent experiments provides the transcript level, where the initial ratio is set to 100% and error bars are ±1 SD for the two independent experiments. Arrows at the bottom indicate nutrient deprivation (red arrow, –) and reintroduction (green arrow, +).
Fig. 5.
Activity–occupancy analysis of the Pol III machinery at RPR1 in response to nutrient availability. (A) Relative transcript levels of RPR1 determined by Northern analysis of total RNA from strain YBC1846 (similar results were obtained with untagged wild-type strain FT4; data not shown), using a probe to the mature form of RPR1 to detect both the unspliced and spliced forms. A probe to Pol II-transcribed U4 serves as a control. (B) Occupancy of RPR1 and analysis of the corresponding transcript in response to nutrient deprivation. The initial occupancy levels of members of the Pol III machinery in rich medium (T = 0 in the regimen) are as follows: Rpc82, 9.3-fold; Brf1, 18-fold; Tfc1, 7.1-fold. These ratios are set to 100% in the graph. The data are treated similarly to that in Fig. 4_D_, except the experimental target amplicon is for RPR1, which includes a large part of the transcribed region. The control amplicon is within the Pol II-transcribed CDC2 ORF.
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