The role of upstream activating factor in suppressing Pol II rRNA transcription in Saccharomyces cerevisiae

Date
1/8/2020
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Abstract

Ribosome synthesis is the most resource and energy – intensive process in all eukaryotic cells and is tightly coupled with growth rate. In addition, defects in synthesis and assembly of ribosomal RNA (rRNA) and ribosomal proteins result in G1 arrest and cell death (Bernstein & Baserga, 2004). As the rate limiting step in ribosome synthesis, rRNA transcription is tightly regulated on many levels. RNA polymerase (Pol I) transcribes the ribosomal DNA (rDNA) to generate a 35S ribosomal RNA (rRNA) precursor which is post-transcriptionally modified to mature 18S, 5.8S, 28S rRNAs (Warner, 1999). However, under chronic stress conditions when Pol I transcription is repressed, rRNA can also be synthesized by RNA polymerase II (Pol II) using a cryptic promoter overlapping the Pol I promoter. This phenomenon of rRNA synthesis by Pol II is termed as ‘polymerase switch’ (Conrad-Webb & Butow, 1995). Since this process is conserved throughout eukaryotes including humans and plants, this phenomenon may play a universal role in the regulation of rRNA. Because the Pol I transcription factor, upstream activating factor (UAF), is known to generate rDNA chromatin inhibitory to Pol II during non-stress conditions, we hypothesized that UAF inhibited the polymerase switch during normal nitrogen conditions and that this inhibition is released during nitrogen deprivation, facilitating the switch. During nitrogen deprivation, UAF steady state levels decreased 2-fold and UAF binding to the rDNA promoter also decreased. Consistent with our hypothesis, UAF subunits H3 and H4 are differentially modified upon nitrogen deprivation with an increase in H3K4 and H3K36 methylation and a decrease in acetylation at H4K5. Contributing to the inhibitory chromatin structure in non-stress conditions, Pol I interacting protein Hmo1 represses polymerase switch as determined by reporter gene assays; whereas, Sir2 does not influence the polymerase switch. Furthermore, transcriptional repressors binding to the Pol II rDNA promoter recruit Ssn6-Tup1 to further repress the Pol II mediated transcription. Thus, during non-stress conditions, UAF triggers the assembly of Pol II inhibitory chromatin and recruitment of HmoI. This inhibitory chromatin is enhanced by the recruitment of the Ssn6-Tup1 repressor. This work has enhanced our understanding of the Pol I regulation during stress conditions and the role Pol II rRNA synthesis plays in overall regulation of ribosome synthesis upon stress.

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Keywords
Stress, Yeast, rRNA
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