Ribosome synthesis is directly linked to the rates of cell growth and proliferation. Transcription of the ribosomal DNA (rDNA) by RNA polymerase I (Pol I) is the first, rate-limiting step in ribosome assembly. In rapidly growing cells, transcription by Pol I accounts for more than 60% of all cellular transcription. The overall hypothesis of this project is that Pol I has evolved sevral unique features that support efficient, robust activity and these features are logical targets for selective inhibition of ribosome synthesis. To test this hypothesis and expand the understanding of ribosome synthesis in eukaryotic cells, this project will focus on three key aspects of rDNA transcription: 1) enzymatic features of Pol I 2) trans-acting factors that control Pol I and 3) sequences in the rDNA that affect transcription elongation and rRNA processing. Pol I has evolved enzymatic properties that suit its cellular role. For example, recent data suggest that the rate-limiting steps in Pol I versus Pol II transcription elongation may differ. These differences could serve as targets for selective inhibition of ribosome biosynthesis. This project will employ rapid mixing kinetic measurements and modeling of resulting data sets to quantitatively describe Pol I transcription elongation. Additional studies will define the elemental steps in transcription elongation that are influenced by specific subunits or domains of the RNA polymerase. Pol I transcription requires trans-acting factors to support the rate of transcription observed in vivo. To test this hypothesis, contributions of three factors to Pol I activity will be characterized in ivo and in vitro. These three factors associate with the rDNA in vivo and have been implicated previously in transcription by Pol II. This study will identify new roles for evolutionary conserve transcription factors in ribosome biogenesis. Transcription elongation by Pol I is functionally coupled to rRNA processing. Sequence elements in the rDNA may directly influence transcription elongation efficiency and nascent rRNA processing. To test this hypothesis, these elements will be identified using single-turnover elongation rate assays in vitro and sequencing of native- elongating transcripts isolated from growing cells. Mutation of identified pause sites and characterization of ribosome assembly will determine the role of these sequences in rRNA processing.
This aim will identify the contribution of DNA elements to the observed orchestration of transcription and rRNA processing. In eukaryotes, several RNA processing events are thought to occur during transcription and this system may serve as a model for the study of these complicated co-transcriptional processes. Transcription by Pol I is a validated target for inhibition of cancer cell proliferation. In order to control cell proliferation via inhibition of Pl I, a detailed understanding of the unique features of rDNA transcription must be established. This study will lay the foundation for ongoing and future projects aimed at selective inhibition of Pol for cancer chemotherapy.
Transcription of the ribosomal DNA by RNA polymerase I is the first, rate-limiting step in ribosome biogenesis. The overall goal of this study is to learn how enzymatic features of RNA polymerase I, trans-acting factors and sequences in the ribosomal DNA template influence the efficiency and regulation of ribosome synthesis. Successful completion of this study will increase our understanding of this central feature of cell biology and inform ongoing and future studies aimed at selective inhibition of RNA polymerase I activity for cancer chemotherapy.
|Clarke, Andrew M; Engel, Krysta L; Giles, Keith E et al. (2018) NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I. Proc Natl Acad Sci U S A 115:E11633-E11641|
|Appling, Francis D; Scull, Catherine E; Lucius, Aaron L et al. (2018) The A12.2 Subunit Is an Intrinsic Destabilizer of the RNA Polymerase I Elongation Complex. Biophys J 114:2507-2515|
|Wei, Ting; Najmi, Saman M; Liu, Hester et al. (2018) Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint. Cell Rep 23:404-414|
|Ucuncuoglu, S; Schneider, D A; Weeks, E R et al. (2017) Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics. Methods Enzymol 582:415-435|
|Zhang, Yinfeng; Najmi, Saman M; Schneider, David A (2017) Transcription factors that influence RNA polymerases I and II: To what extent is mechanism of action conserved? Biochim Biophys Acta Gene Regul Mech 1860:246-255|
|Appling, Francis D; Lucius, Aaron L; Schneider, David A (2017) Quantifying the influence of 5'-RNA modifications on RNA polymerase I activity. Biophys Chem 230:84-88|
|Cornelison, Robert; Dobbin, Zachary C; Katre, Ashwini A et al. (2017) Targeting RNA-Polymerase I in Both Chemosensitive and Chemoresistant Populations in Epithelial Ovarian Cancer. Clin Cancer Res 23:6529-6540|
|Zhang, Yinfeng; French, Sarah L; Beyer, Ann L et al. (2016) The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I. J Biol Chem 291:3010-8|
|Ucuncuoglu, Suleyman; Engel, Krysta L; Purohit, Prashant K et al. (2016) Direct Characterization of Transcription Elongation by RNA Polymerase I. PLoS One 11:e0159527|
|Engel, Krysta L; French, Sarah L; Viktorovskaya, Olga V et al. (2015) Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I. Mol Cell Biol 35:2321-31|
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