Specific DNA features of the RNA polymerase I core promoter element targeted by core factor.

DNA binding preferences of S. cerevisiae RNA polymerase I Core Factor reveal a preference for the GC-minor groove and a conserved binding mechanism

In Saccharomyces cerevisiae, Core Factor (CF) is a key evolutionary conserved transcription initiation factor that helps recruit RNA polymerase I (Pol I) to the ribosomal DNA (rDNA) promoter. Upregulated Pol I transcription has been linked to many cancers, and targeting Pol I is an attractive and emerging anti‐cancer strategy. Using the yeast model system, we characterized how CF binds to the Pol I promoter by electrophoretic mobility shift assays (EMSA). Synthetic DNA competitors along with anti‐tumor drugs and nucleic acid stains that act as DNA groove blockers were used to discover structural characteristics of CF binding. Our results show that CF employs a unique mechanism where it prefers the GC‐rich minor groove of the rDNA promoter, which may offer a new avenue to more specifically target upregulated Pol I activity in cancer.Support or Funding InformationThe work was supported by B.A.K. grants from the US National Institutes of Health (NIH) NCI (5K22CA184235), a Sinsheimer Scholar award from the Alexandrine and Alexander L. Sinsheimer Fund, Central New York Community Foundation, Joseph C. George Fund, and Virginia Simons & Dr. C. Adele Brown Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A central component in S. cerevisiae RNA polymerase I (Pol I) pre‐initiation complex (PIC) formation and transcriptional regulation is the evolutionarily conserved initiation factor termed Core Factor (CF). CF is a heterotrimer composed of Rrn6, Rrn11, and the TFIIB‐related factor Rrn7. Recent Cryo‐EM structures of the Pol I initiation complex reveal that CF resembles a right hand grasping promoter DNA in which key contacts are mediated by Rrn11 and Rrn7 (Han et al., eLife, 2017). Similar to its Pol II counterpart TFIIB, CF subunit Rrn7 consists of an N‐terminal zinc ribbon (ZR) domain followed by a linker region and two cyclin fold repeats. We systematically mutagenized the Rrn7 ZR and linker domains to further understand their role in Pol I transcription initiation. We found that ZR and linker mutants are still able to form the CF complex and assemble a PIC, yet are defective in promoter opening. These findings, paired with the Cryo‐EM structure, suggest that CF acts as a ratchet to drive promoter DNA into the active site of Pol I and that the Rrn7 ZR and linker domains play a dynamic role in ATP‐independent promoter opening.Support or Funding InformationThis work was supported by grants awarded to BAK: National Cancer Institute (5K22CA184235), Alexandrine and Alexander L. Sinsheimer Scholar award, and SUNY Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I). We have cloned the gene and found that it encodes a protein of 507 amino acids. We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope-tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11. This protein complex complemented rrn6 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF). The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit. The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA-binding protein. Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein. These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein. RRN11 was shown to be an essential gene, and [3H]uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo. Thus all three subunits of CF are essential for rDNA transcription. Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAFI48, TAFI63, and TAFI110. No significant similarity was detected between two sets of the proteins. Similarity as well as differences between CF and SL1 are discussed.

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

RNA Polymerase I(Pol I) is one of three essential DNA dependent RNA polymerases in eukaryotes and is responsible for synthesizing ribosomal RNA. A critical and essential Pol I transcription factor in yeast is Core Factor (CF) which binds to a ~24 bp region in the rDNA promoter called the Core Element (CE). CF plays fundamental roles in the Pol I transcription process helping to recruit Pol I and open Pol I promoter DNA before initiation. Previously, it was not yet known how CF precisely recognized the CE. When interacting with DNA, proteins use two main mechanisms: i) base‐pair readout, and ii) shape/structural readout. Base‐readout is the most common and is dominated by hydrogen bonding between the amino acid residues and base‐pair hydrogen bond donors and acceptors. The second mechanism centers around DNA shape readout where proteins target specific DNA features such as curvature, bendability, and groove width. Previous studies from our lab have shown that CF and its human orthologue, Selectivity Factor 1 (SL1), use an evolutionarily conserved mechanism to target DNA which is governed by interactions with the GC minor groove, a unique surface rarely targeted by DNA‐binding proteins. To further understand the extent of structural recognition as well as specific DNA features CF may be using to interact with CE, we have employed a variety of selection‐based methods to resolve the structural rules governing CF’s interaction with DNA. Our findings are consistent with a model that CF‐CE interaction is governed primarily by DNA shape‐based structural features rather than sequence.

Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position -1 to -2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from -27 to -16. Core Factor's intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from −27 to −16. Core Factor’s intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.DOI:http://dx.doi.org/10.7554/eLife.27414.001

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

Transcription Factors and DNA Play Hide and Seek.

To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.

To grow and to divide, cells are dependent on protein synthesis, and protein synthesis depends on ribosomes. Ribosomes are complex molecular machines that contain four different RNA molecules and 79 different proteins, produced by three different RNA polymerases. RNA polymerase I (Pol I) synthesizes the large ribosomal RNA (rRNA) precursor (35S RNA in yeast), which is then processed into mature 18S, 28S, and 5.8S RNAs; RNA polymerase II (Pol II) synthesizes the mRNAs encoding the ribosomal proteins; and RNA polymerase III (Pol III) synthesizes the small 5S RNA molecule. The biosynthesis of ribosomes by the three RNA polymerases uses an enormous amount of the cell resources. In a yeast cell, rRNA transcription represents ∼60% of total transcription, and transcription of the ribosomal protein mRNAs represents ∼50% of all Pol II transcription initiation events (Warner 1999; Rudra and Warner 2004). Accordingly, ribosome biosynthesis is tightly regulated with cell growth and proliferation. Thus, when the demand for protein synthesis is reduced, as occurs with cells in stationary phase after nutrient deprivation, the production of new ribosomes is greatly reduced. Ribosome assembly requires the coordinated production of ribosome components. On the one hand, equimolar amounts of each ribosome protein must be produced. In yeast, the coregulation of ribosomal protein synthesis is exerted mainly at the level of ribosomal protein gene transcription. On the other hand, sufficient amounts of each ribosomal protein must be produced to allow correct assembly and processing of rRNAs. This suggests that the ribosomal protein and rRNA synthesis machineries are somehow coregulated. Indeed, an essential signal transduction pathway for the modulation of rRNA, ribosomal proteins, and 5S rRNA in response to nutrient availability is the TOR (Target of Rapamycin) kinase pathway. This pathway is activated in the presence of nutrients, and inactivation of TOR—for example, by the antibiotic rapamycin—mimics the effects of starvation (for review, see Crespo and Hall 2002). Rapamycin inhibition of the TOR pathway is known to repress rRNA transcription by Pol I, Pol III transcription, and Pol II transcription of ribosomal protein genes (Zaragoza et al. 1998; Powers and Walter 1999). However, we still know little about how TOR controls ribosome component synthesis, in particular, whether each of the three RNA polymerase machineries is controlled separately, whether one of them is the primary target that then controls the others, or whether several mechanisms of crosstalk ensure coordinated regulation. In this issue of Genes & Development, Laferte et al. (2006) present data suggesting that in yeast, accumulation of large ribosomal RNAs as a result of deregulated Pol I transcription leads to a corresponding accumulation of ribosomal proteins, 5S rRNA, and fully assembled ribosomes. Thus, increased Pol I transcription activity can orchestrate the coordinated increased accumulation of all ribosomal components, suggesting a central role for RNA polymerase I activity in the coordination of ribosomal component synthesis. Transcription by Pol I in yeast is known to depend on four factors: Three of them—TBP (TATA-box binding protein) and the multisubunit complexes UAF (upstream activating factor) and CF (core factor)—bind together to rDNA promoters; the fourth, Rrn3, associates reversibly with Pol I and renders it competent for transcription initiation, at least in part by bridging Pol I and the promoter-bound factors (for review, see Moss and Stefanovsky 2002; Grummt 2003; Russell and Zomerdijk 2006). The association of Rrn3 with Pol I controls the activity of the enzyme, and the percentage of active Pol I in the cell appears in turn to play a determinant role in the control of rRNA transcription. The investigators created a yeast strain, which they named CARA (for Constitutive Association of Rrn3 and A43), in which the endogenous genes coding for Rrn3 and A43, the Pol I subunit with which Rrn3 is known to associate (Peyroche et al. 2000), were deleted. Rrn3 and A43 activities were supplied as a fusion protein expressed from a 2-μ plasmid. The fusion protein assembled properly with the other Pol I subunits to form a constitutively active enzyme. Remarkably, under normal growth conditions, the CARA strain behaves like the wild type: It has the same doubling time and the same amount of ribosomal particles, and it has a similar mRNA expression pattern. Thus, the CARA strain has a normal physiology. Modulation of Pol I transcription through TOR targets Rrn3. Upon rapamycin treatment, Rrn3 dissociates from the Pol I complex, resulting in an arrest of transcription Corresponding author. E-MAIL Nouria.Hernandez@unil.ch; FAX 41-21-692-39-25. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1460706.