Structural basis of nucleosome deacetylation and DNA linker tightening by Rpd3S histone deacetylase complex.

In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP. These structures suggest that Rpd3S utilizes a conserved Sin3 basic surface to navigate through the nucleosomal DNA, guided by its interactions with H3K36 methylation and the extra-nucleosomal DNA linkers, to target acetylated H3K9 and sample other histone tails. Furthermore, our structures illustrate that Rpd3S reconfigures the DNA linkers and acts in concert with Hho1 to engage the NCP, potentially unraveling how Rpd3S and Hho1 work in tandem for gene silencing.

Vibrio parahaemolyticus prey targeting requires autoproteolysis-triggered dimerization of the type VI secretion system effector RhsP.

The rearrangement hotspot (Rhs) repeat is an ancient giant protein fold found in all domains of life. Rhs proteins are polymorphic toxins that could either be deployed as an ABC complex or via a type VI secretion system (T6SS) in interbacterial competitions. To explore the mechanism of T6SS-delivered Rhs toxins, we used the gastroenteritis-associated Vibrio parahaemolyticus as a model organism and identified an Rhs toxin-immunity pair, RhsP-RhsPI. Our data show that RhsP-dependent prey targeting by V. parahaemolyticus requires T6SS2. RhsP can bind to VgrG2 independently without a chaperone and spontaneously self-cleaves into three fragments. The toxic C-terminal fragment (RhsPC) can bind to VgrG2 via a VgrG2-interacting region (VIR). Our electron microscopy (EM) analysis reveals that the VIR is encapsulated inside the Rhs ß barrel structure and that autoproteolysis triggers a dramatic conformational change of the VIR. This alternative VIR conformation promotes RhsP dimerization, which significantly contributes to T6SS2-mediated prey targeting by V. parahaemolyticus.

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins.

This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.