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.