Transcription of transfer RNA (tRNA) genes by RNA polymerase (Pol) III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) to reveal structural and real-time kinetic insights into how the 520-kDa yeast TFIIIC complex engages A-box and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of τA and τB subcomplexes bound to the A-box and B-box were obtained at 3.7 and 2.5 Å resolution, respectively, while cryo-EM single-particle mapping determined the specific distance and relative orientation of the τA and τB subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of τA and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

Centriole integrity, vital for cilia formation and chromosome segregation, is crucial for human health. The inner scaffold within the centriole lumen composed of the proteins POC1B, POC5 and FAM161A is key to this integrity. Here, we provide an understanding of the function of inner scaffold proteins. We demonstrate the importance of an interaction network organised by POC1A-POC1B heterodimers within the centriole lumen, where the WD40 domain of POC1B localises close to the centriole wall, while the POC5-interacting WD40 of POC1A resides in the centriole lumen. The POC1A-POC5 interaction and POC5 tetramerization are essential for inner scaffold formation and centriole stability. The microtubule binding proteins FAM161A and MDM1 by binding to POC1A-POC1B, likely positioning the POC5 tetramer near the centriole wall. Disruption of POC1A or POC1B leads to centriole microtubule defects and deletion of both genes causes centriole disintegration. These findings provide insights into organisation and function of the inner scaffold. The inner scaffold is key for centriole stability but its organization is unclear. Here, the authors show the formation of POC1A-POC1B heterodimers, where POC1B resides near the centriole wall and POC1A associates with a POC5 tetramer/Centrin complex.

Loss of H2A–H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5′-triphosphate–dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A–H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3–H4 histone interface activates INO80, independently of the H2A–H2B acidic patch. Our findings reveal how the loss of H2A–H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.

Transcription factor (TF) IIIC recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in transfer RNA (tRNA) genes by TFIIIC modules τA and τB is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo–electron microscopy structures of the six-subunit human TFIIIC complex unbound and bound to a tRNA gene. The τB module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both τA and τB connecting the two subcomplexes via a ~550–amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.

In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA-nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNA(Pro) in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.

Co(III)(tacn) and Ga(III)(tacn) fragments (tacn = 1,4,7-triazacyclononane) are suitable metal probes for the detection of the tridentate chelating sites of a glycose. Three moles of hydroxide per mole of cobalt or gallium support triple deprotonation of the chelating triol functions at a glycose's backbone. The individual chelating sites are detected using 1D and 2D NMR techniques. The metal-binding sites always include the hydroxy function at the anomeric carbon atom. Chelators are derived from both the pyranose and the furanose isomers while forming five- and six-membered chelate rings by the use of cis,cis-1,2,3-triol functions. The assignment of less frequently occurring ligand isomers are supported by a DFT approach. Crystal-structure analysis on Na(4)[Cr(beta-d-Manf1,2,3H(-3))(2)]NO(3).8.5H(2)O (Man = mannose) additionally upholds the NMR assignments.