Structural Synergy and Molecular Crosstalk between Bacterial Helicase Loaders and Replication Initiators

SummaryThe loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from Aquifex aeolicus to 2.7 Å resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. Using X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core, together with cross-linking coupled to mass spectrometry, we were able to use IMP to position and orient all eIF3 components on the 40S•eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. For more information about how to reproduce this modeling, see https://salilab.org/40S-eIF1-eIF3 or the README file

A comprehensive landscape of 60S ribosome biogenesis factors

Eukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryoelectron microscopy (cryo-EM) maps have defined the molecular interactions of RBFs during maturation, but many transient and dynamic interactions, particularly during early assembly, remain uncharacterized. Using quantitative proteomics and crosslinking coupled to mass spectrometry (XL-MS) data from an extensive set of pre-ribosomal particles, we derive a comprehensive and time-resolved interaction map of RBF engagement during 60S maturation. We localize 22 previously unmapped RBFs to specific biogenesis intermediates and validate our results by mapping the catalytic activity of the methyltransferases Bmt2 and Rcm1 to their predicted nucleolar 60S intermediates. Our analysis reveals the interaction sites for the RBFs Noc2 and Ecm1 and elucidates the interaction map and timing of 60S engagement by the DEAD-box ATPases Dbp9 and Dbp10. Our data provide a powerful resource for future studies of 60S ribosome biogenesis.publishe

Nucleotide and Partner-Protein Control of Bacterial Replicative Helicase Structure and Function

Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors

Architecture of the large subunit of the mammalian mitochondrial ribosome

ISSN:0028-0836ISSN:1476-468