Eukaryotes express three or more multisubunit nuclear RNA polymerases (Pols) referred to as Pols I, II, and III, each of which synthesizes a specific subset of RNAs. Consistent with the diversity of their target genes, eukaryotic cells have evolved divergent cohorts of transcription factors and enzymatic properties for each RNA polymerase system. Over the years, many trans-acting factors that orchestrate transcription by the individual Pols have been described; however, little effort has been devoted to characterizing the molecular mechanisms of Pol I activity. To begin to address this gap in our understanding of eukaryotic gene expression, here we establish transient-state kinetic approaches to characterize the nucleotide incorporation mechanism of Pol I. We collected time courses for single turnover nucleotide incorporation reactions over a range of substrate ATP concentrations that provide information on both Pol I's nucleotide addition and nuclease activities. The data were analyzed by model-independent and model-dependent approaches, resulting in, to our knowledge, the first minimal model for the nucleotide addition pathway for Pol I. Using a grid searching approach we provide rigorous bounds on estimated values of the individual elementary rate constants within the proposed model. This work reports the most detailed analysis of Pol I mechanism to date. Furthermore, in addition to their use in transient state kinetic analyses, the computational approaches applied here are broadly applicable to global optimization problems.
Spt5 is a transcription factor conserved in all three domains of life. Spt5 homologues from bacteria and archaea bind the largest subunit of their respective RNA polymerases. Here we demonstrate that Spt5 directly associates with RNA polymerase (Pol) I and RNA Pol II in yeast through its central region containing conserved NusG N-terminal homology and KOW domains. Deletion analysis of SPT5 supports our biochemical data, demonstrating the importance of the KOW domains in Spt5 function. Far Western blot analysis implicates A190 of Pol I as well as Rpb1 of Pol II in binding Spt5. Three additional subunits of Pol I may also participate in this interaction. One of these subunits, A49, has known roles in transcription elongation by Pol I. Interestingly, spt5 truncation mutations suppress the cold-sensitive phenotype of rpa49⌬ strain, which lacks the A49 subunit in the Pol I complex. Finally, we observed that Spt5 directly binds to an essential Pol I transcription initiation factor, Rrn3, and to the ribosomal RNA. Based on these data, we propose a model in which Spt5 is recruited to the rDNA early in transcription and propose that it plays an important role in ribosomal RNA synthesis through direct binding to the Pol I complex.The study of ribosome biogenesis is fundamentally important. Multiple studies have connected dysregulation of the factors that affect ribosome biogenesis with cell transformation and cancer (1, 2). Transcription of ribosomal DNA (rDNA) 2 by RNA polymerase (Pol) I is the initial step of ribosome synthesis. In Saccharomyces cerevisiae (for simplicity referred to as "yeast"), Pol I produces the 35S rRNA precursor, which is cotranscriptionally and post-transcriptionally processed into the mature 18, 5.8, and 25 S rRNA species that form the bulk of the small and large ribosomal subunits.Pol I consists of 14 subunits; seven of which are Pol I-specific, whereas the other seven are shared with RNA Pol II and/or RNA Pol III. Pol I transcription initiation in vivo requires four additional factors: TATA-binding protein, Rrn3, core factor, and the upstream activating factor. Formation of the Rrn3-Pol I complex is a crucial step in the recruitment of Pol I to the promoter. As such, this step in transcription is tightly regulated. However, the initiation step alone cannot account for the magnitude of regulation of Pol I transcription observed in vivo (3,4). Recent data demonstrate that transcription elongation by Pol I is also regulated and that transcription elongation by Pol I is functionally coupled to rRNA processing (5-7). Thus, factors that influence the elongation step in transcription may play one or more important roles in overall ribosome synthesis.Although Pol I transcription elongation remains understudied, significantly more is known about factors that affect Pol II transcription elongation. One of these factors is the conserved Spt4/5 complex. In mammalian and Drosophila cells, Spt4/5 induces pausing of the Pol II elongation complex in close proximity to the promoter (reviewed in Ref. 8). This pa...
Transcription of ribosomal DNA by RNA polymerase I is a central feature of eukaryotic ribosome biogenesis. Since ribosome synthesis is closely linked to cell proliferation, there is a need to define the molecular mechanisms that control transcription by RNA polymerase I. To fully define the factors that control RNA polymerase I activity, biochemical analyses using purified transcription factors are essential. Although such assays exist, one limitation is the low abundance and difficult purification strategies required for some of the essential transcription factors for RNA polymerase I. Here, we describe a new method for expression and purification of the three subunit core factor complex from Escherichia coli. We demonstrate that the recombinant material is more active than yeast-derived core factor in assays for RNA polymerase I transcription in vitro. Finally, we use recombinant core factor to differentiate between two opposing models for the role of the TATA-binding protein in transcription by RNA polymerase I.
All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called "cleavage factors" appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase's cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase-cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases.
Despite sharing a highly conserved core architecture with their prokaryotic counterparts, eukaryotic multisubunit RNA polymerases (Pols) have undergone structural divergence and biological specialization. Interesting examples of structural divergence are the A12.2 and C11 subunits of Pols I and III, respectively. Whereas all known cellular Pols possess cognate protein factors that stimulate cleavage of the nascent RNA, Pols I and III have incorporated their cleavage factors as bona fide subunits. Although it is not yet clear why these polymerases have incorporated their cleavage factors as subunits, a picture is emerging that identifies roles for these subunits beyond providing nucleolytic activity. Specifically, it appears that both A12.2 and C11 are required for efficient termination of transcription by Pols I and III. Given that termination involves destabilization of the elongation complex (EC), we tested whether A12.2 influences stability of the Pol I EC. Using, to our knowledge, a novel assay to measure EC dissociation kinetics, we have determined that A12.2 is an intrinsic destabilizer of the Pol I EC. In addition, the salt concentration dependence of Pol I EC dissociation kinetics suggests that A12.2 alters electrostatic interactions within the EC. Importantly, these data present a mechanistic basis for the requirement of A12.2 in Pol I termination. Combined with recent work demonstrating the direct involvement of A12.2 in Pol I nucleotide incorporation, this study further supports the concept that A12.2 cannot be viewed solely as a cleavage factor.
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