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RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination.

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RNA polymerase I (Pol I) synthesizes the majority of the ribosomal RNA (rRNA) in a eukaryotic cell, which is then assembled into the mature ribosome with ribosomal proteins. Pol I transcribes a single gene, the 35S gene in yeast, containing three gene elements (18S, 5.8S, and 25S) and four spacer regions. Previous literature demonstrates that processing events occur both co‐ and post‐transcriptionally so that the spacer regions are removed and degraded, resulting in mature 18S, 5.8S, and 25S rRNAs. While it has been established that rRNA processing begins during transcription, the relationship between Pol I transcription elongation properties and processing events have not been well defined. The objective of this project was to determine whether transcription elongation kinetics have a regulatory role in rRNA processing, and we hypothesized that if transcription elongation was impaired, this would result in defects in processing. To test this hypothesis, we generated a yeast strain containing a point mutation in RPA190, the largest subunit of Pol I. We found that there was a modest decrease in transcription elongation rate and an increase in pausing in the mutant compared to wild‐type Pol I in vitro. To analyze elongation effects of the mutant Pol I in vivo, we used native elongating transcript sequencing, which probes for Pol I occupancy on the ribosomal DNA template at single‐nucleotide resolution. Our in vivo results corroborated the in vitro findings, indicating that this mutation introduced an increase in pausing across the template during transcription elongation. Furthermore, Northern blots and polysome profiling indicated that rRNA processing and ribosome assembly were impaired when this mutation was introduced. These three independent in vitroand in vivo experimental strategies converge to demonstrate that transcription elongation and Pol I pausing may play a regulatory role in downstream processing steps.