Abstract
The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.
Highlights
The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined
Native elongating transcript sequencing (NET-seq) adapted for Saccharomyces cerevisiae Pol I elucidated that Pol I occupancy on ribosomal DNA is heterogeneous, which could imply that DNA sequence impacts pausing and Pol I elongation rate in vivo [9]
These findings demonstrate that Pol I physically interrogates the downstream DNA sequence and that these sequence elements directly influence rRNA synthesis both in vivo and in vitro
Summary
I with a preannealed RNA:DNA hybrid (A), and nontemplate DNA is added to form the elongation complex bubble (B). The actual size of the bubble has not been determined for Pol I with these templates, we do not depict annealing of the upstream or downstream regions of the DNA. NET-seq revealed that this downstream DNA sequence effect is conserved in vivo. Together, these findings demonstrate that Pol I physically interrogates the downstream DNA sequence (at least in part through the A12 subunit) and that these sequence elements directly influence rRNA synthesis both in vivo and in vitro
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