Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I

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) fromSaccharomyces cerevisiaehas not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausingin vitroin response to downstream DNA.In vitrostudies 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 inSaccharomyces cerevisiaesuggests that these downstream sequence elements influence Pol Iin 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 bothin vivoandin vitro.