The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.The project was supported by grant BFU2013-48374-P of the Spanish MINECO and by the Ramón Areces Foundation. O.G. held a research contract under the Ramón y Cajal program of the Spanish MINECO (RYC-2011-07967). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish MINECO.Peer reviewe

Cryo-EM Structures of Free Monomeric and Rrn3-bound RNA Polymerase I Unveil the Structural Changes in the Transition from Inactive Dimers to the Activated State

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 29-03-201

Cryo-em structures of free monomeric and RRN3-bound RNA polynerase I unveil the structural changes in the transition fom inactive dimers to the activated state

127 p.-18 fig.-17 tab.In eukaryotes, DNA polymerase I is the kilodalton enzyme responsible for ribosonal DNA (rDNA) transcription, leading to the synthesis of the ribosonal RNA precusor. Pol I's activity is crucial for ribosome biogenesis and therefore, its modulation influences cell growth. The intiation factor rDNA, conseved in eukaryotes, binds Pol I corresponds to an activated state of the enzyme, whose activity can be regulated by assembling or disrupting Pol I:Rm3. the cryo-electron microscopy (cryo-EM) structures of yeast free monomeric Pol I at 4.9 A resolution and Pol I in complex with Rm3 at 7.7 A are the major findings reported here. The derived pseudo-atomic models unveil the structural changes in the transition from inactive Pol I dimers. previously solved by X-ray crystalography, to free monovers and from these to the activated state bound to Rm.3 In addition, analytical ultracentrifugation suggets that yeast Rm3 dimers found in solution migth establish a dimer-monover equilibrium in vitro upon dilution. Also, electrophoretic mobility shift assays indicate yeast Rm3 could not bind DNA as previously described for the mammalian homolog. These resuts provide valuable information on Pol I and Rn3 changes required for enzyme activation.Beca FPI BES-2011-044359 del Ministerio de Economía Industria y CompetitividadPeer reviewe

Sub1, two domains, two functions: transcription initiation versus elongation

Póster presentado a la EMBO Conferences: Gene Transcription in Yeast: From regulatory networks to mechanisms, celebrada en Sant Feliu de Guixols (España) del 14 al 19 de junio de 2014.We have performed a detailed analysis of the ssDNA binding and C-terminal domains of Sub1 from S. cerevisiae and identified the essential residues involved in Sub1 DNA binding capacity, which are not required during transcription elongation. Additionally, our data indicates that the C-terminus domain of Sub1, the function of which was previously unknown, might be important in promoting Sub1 release from the promoter to facilitate transcription elongation. In summary, Sub1 plays a dual function during transcription, being a pre-initiation factor via its DNA binding domain, and a transcription elongation factor through its C-terminus domain.This work was funded by the Ministerio de Economía y Competitividad.Peer Reviewe

Sub1 contacts the RNA polymerase II stalk to modulate mRNA synthesis

Biogenesis ofmessenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II(RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain,Rpb4/7, likely through its C-terminal region,and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2013-48374-P]; Predoctoral fellowships from MINECO (to J.A.L.); Technician Formation Program from the Spanish National Research Council (CSIC) (to N.G.-P.). Funding for open access charge: MINECO [BFU2013-48374-P].Peer reviewe

Sub1/PC4, more than a RNAPII transcription factor

Trabajo presentado en la II Reunión de la Red de Excelencia Temática: RNA Life, celebrada en Madrid (España), los días 19 y 20 de julio de 2017Biogenesis of mRNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions, and Sub1 globally influences CTD phosphorylation, though its mechanism remained mostly unknown until recently. Sub1 was initially identified as a coactivator factor, with a role during transcription initiation, due to conserved functional and structural features with human PC4. However, over the last years many evidences showed that it influences processes downstream during mRNA biogenesis, such as elongation, termination and RNAPII phosphorylation. The recent discover that Sub1 directly interacts with the RNAPII stalk adds new insights into how it achieves all these tasks. Moreover, we showed that this interaction likely occurs via the carboxi-terminal region of Sub1 (Sub1-CT), of unknown function so far. We also demonstrated that a complete RNAPII-CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Altogether, our results indicate that Sub1 in association with Rpb4/7 and the CTD phosphatase Fcp1, modulates CTD phosphorylation, which crucially regulates the biogenesis of mRNAs. We also provide evidence indicating that Sub1 contributes to RNAPII clamp function elucidating Sub1 role during the transition from the open to the closed complex formation, thus facilitating transcription elongation. On the other hand, genome wide studies showed that Sub1 also binds to all RNAP III-transcribed genes and the rDNA gene transcribed by RNAPI, though, in this latter case, Sub1 binding is controversial. In agreement, genome wide experiments performed by us show that tRNAs transcription is decreased when lacking SUB1. As in the case of RNAPII, Sub1 works in different steps of RNAPIII transcription cycle. It stimulates both transcription initiation and reinitiation in vitro. Additionally, Sub1 and PC4, as ssDNA binding proteins, have been involved in the maintenance of genome stability. Therefore, it is clear that the role of Sub1 in transcription regulation in particular, and in gene expression in general, is more complicated and important than anticipated. Although many progresses have been done regarding Sub1 role in transcription, several important questions remains unanswered: What is the role of Sub1-CT? Is Sub1 a gene looping factor? Is Sub1 shared by all the three RNAPs?Peer reviewe

Nucleosome dyad determines the H1 C-terminus collapse on distinct DNA arms

© 2023 Elsevier LtdNucleosomes are symmetric structures. However, binding of linker histones generates an inherently asymmetric H1-nucleosome complex, and whether this asymmetry is transmitted to the overall nucleosome structure, and therefore also to chromatin, is unclear. Efforts to investigate potential asymmetry due to H1s have been hampered by the DNA sequence, which naturally differs in each gyre. To overcome this issue, we designed and analyzed by cryo-EM a nucleosome reconstituted with a palindromic (601L) 197-bp DNA. As in the non-palindromic 601 sequence, H1 restricts linker DNA flexibility but reveals partial asymmetrical unwrapping. However, in contrast to the non-palindromic nucleosome, in the palindromic nucleosome H1 CTD collapses to the proximal linker. Molecular dynamics simulations show that this could be dictated by a slightly tilted orientation of the globular domain (GD) of H1, which could be linked to the DNA sequence of the nucleosome dyad