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

Nucleosomes 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.

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.

Sub1 contacts the RNA polymerase II stalk to modulate mRNA synthesis.

Biogenesis of messenger 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.