Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y.,

RNA polymerase I (Pol I) is the most specialized eukaryotic Pol. It is only responsible for the synthesis of pre-ribosomal RNA (rRNA), the precursor of 18S, 5.8S and 28S rRNA, the most abundant cellular RNA types. Aberrant Pol I transcription is observed in a wide variety of cancers and its down-regulation is associated with several genetic disorders. The regulation and mechanism of Pol I transcription is increasing in clarity given the numerous high-resolution Pol I structures that have helped bridge seminal genetic and biochemical findings in the field. Here, we review the multifunctional roles of an important TFIIF- and TFIIE-like subcomplex composed of the Pol I subunits A34.5 and A49 in yeast, and PAF49 and PAF53 in mammals. Recent analyses have revealed a dynamic interplay between this subcomplex at nearly every step of the Pol I transcription cycle in addition to new roles in chromatin traversal and the existence of a new helix-turn-helix (HTH) within the A49/PAF53 linker domain that expands its dynamic functions during the Pol I transcription process.

Our knowledge of the mechanism of rDNA transcription has benefitted from the combined application of genetic techniques in yeast, and progress on the biochemistry of the various components of yeast rDNA transcription. Nomura’s laboratory derived a system in yeast for screening for mutants essential for ribosome biogenesis. Such systems have allowed investigators to not only determine if a gene was essential, but to analyze domains of the proteins for different functions in rDNA transcription in vivo. However, because there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an inducible degron that allows us to combine a “genetics-like” approach to studying mammalian rDNA transcription with biochemistry. Using this system, we show that the mammalian homologue of yeast A49, PAF53, is required for rDNA transcription and mitotic growth. Further, we have been able to study the domains of the protein required for activity. We have found that while the C-terminal, DNA-binding domain (tWH) was necessary for complete function, the heterodimerization and linker domains were also essential. Analysis of the linker identified a putative DNA-binding domain. We have confirmed that the helix-turn-helix (HTH) of the linker constitutes a second DNA-binding domain within PAF53 and that the HTH is essential for PAF53 function.

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different.

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