Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A.

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd(2+)) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd(2+) rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd(2+), but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I-Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag(+) and Hg(2+), which likewise perturb the Pol I-Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I-Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals.

Odd RNA polymerases or the A(B)C of eukaryotic transcription.

Pioneering studies on eukaryotic transcription were undertaken with the bacterial system in mind. Will the bacterial paradigm apply to eukaryotes? Are there promoter sites scattered in the eukaryotic genome, and sigma-like proteins? Why three forms of RNA polymerase in eukaryotic cells? Why are they structurally so complex, in particular RNA polymerases I and III, compared to the bacterial enzyme? These questions and others that were raised along the way are evoked in this short historical survey of odd RNA polymerases studies, with some emphasis on the contribution of these studies to our global understanding of eukaryotic transcription systems. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription.

We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10(3) over the error rate determined solely by selectivity (1.8 x 10(-4)). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.

Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III.

RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle analysis, reveals a hand-like shape typical of RNA polymerases. Compared to RNAPII, RNAPIII is characterized by a bulkier stalk and by prominent features extending from the DNA binding cleft. We attribute the latter primarily to five RNAPIII-specific subunits, present as two distinct subcomplexes (C82/C34/C31 and C53/C37). Antibody labeling experiments localize the C82/C34/C31 subcomplex to the clamp side of the DNA binding cleft, consistent with its known role in transcription initiation. The C53/C37 subcomplex appears to be situated across the cleft, near the presumed location of downstream DNA, accounting for its role in transcription termination. Our structure rationalizes available mutagenesis and biochemical data and provides insights into RNAPIII-mediated transcription.

Is ribosome synthesis controlled by pol I transcription?

Regulation of growth ultimately depends on the control of synthesis of new ribosomes. Ribosome biogenesis is thus a key element of cell biology, which is tightly regulated in response to environmental conditions. In eukaryotic cells, the supply of ribosomal components involves the activities of the three forms of nuclear RNA polymerase (Pol I, Pol II and Pol III). Recently, we demonstrated that upon rapamycin treatment, a partial derepression of Pol I transcription led to a concomitant derepression of Pol II transcription restricted to a small subset of class II genes encompassing the genes encoding all ribosomal proteins, and 19 additional genes. The products of 14 of these 19 genes are principally involved in rDNA structure, ribosome biogenesis or translation, whereas the five remaining genes code for hypothetical proteins. We demonstrate that the proteins encoded by these five genes are required for optimal pre-rRNA processing. In addition, we show that cells in which regulation of Pol I transcription was specifically impaired are either resistant or hypersensitive to different stresses compared to wild-type cells. These results highlight the critical role of the regulation of Pol I activity for the physiology of the cells.

The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components.

Regulation of ribosome biogenesis is a key element of cell biology, not only because ribosomes are directly required for growth, but also because ribosome production monopolizes nearly 80% of the global transcriptional activity in rapidly growing yeast cells. These observations underscore the need for a tight regulation of ribosome synthesis in response to environmental conditions. In eukaryotic cells, ribosome synthesis involves the activities of the three nuclear RNA polymerases (Pol). Although postulated, there is no clear evidence indicating whether the maintenance of an equimolar supply of ribosomal components reflects communication between the nuclear transcriptional machineries. Here, by constructing a yeast strain expressing a Pol I that remains constitutively competent for the initiation of transcription under stress conditions, we demonstrate that derepression of Pol I transcription leads to a derepression of Pol II transcription that is restricted to the genes encoding ribosomal proteins. Furthermore, we show that the level of 5S rRNA, synthesized by Pol III, is deregulated concomitantly with Pol I transcription. Altogether, these results indicate that a partial derepression of Pol I activity drives an abnormal accumulation of all ribosomal components, highlighting the critical role of the regulation of Pol I activity within the control of ribosome biogenesis.

A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation.

While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIdelta), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37-C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37-C53 complex required the simultaneous addition of C11 to Pol IIIdelta for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation.

CTD kinase I is involved in RNA polymerase I transcription.

RNA polymerase II carboxy terminal domain (CTD) kinases are key elements in the control of mRNA synthesis. Yeast CTD kinase I (CTDK-I), is a non-essential complex involved in the regulation of mRNA synthesis at the level of transcription elongation, pre-mRNA 3' formation and nuclear export. Here, we report that CTDK-I is also involved in ribosomal RNA synthesis. We show that CTDK-I is localized in part in the nucleolus. In its absence, nucleolar structure and RNA polymerase I transcription are affected. In vitro experiments show an impairment of the Pol I transcription machinery. Remarkably, RNA polymerase I co-precipitates from cellular extracts with Ctk1, the kinase subunit of the CTDK-I complex. In vitro analysis further demonstrates a direct interaction between RNA polymerase I and Ctk1. The results suggest that CTDK-I might participate in the regulation of distinct nuclear transcriptional machineries, thus playing a role in the adaptation of the global transcriptional response to growth signalling.

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

The A14-A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4-Rpb7 pol II subunits.

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the omega subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.

Localization of the yeast RNA polymerase I-specific subunits.

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.