Biology subjects

Sedivy, E. L.

Publications and source records attributed to Sedivy, E. L..

3 recordsLinked to original sources

bioRxiv2025

The structure of a thermostable phage's Portal Vertex and Neck Complex illuminates its maturation process

Viruses assemble from component parts inside their host cells, but the mechanisms coordinating this complex process are not completely understood. In tailed bacteriophages, the genome is packaged into its capsid shell through the portal complex. The portal complex then closes to retain DNA and connects to the tail, which is required for host recognition and infection. The trigger to stop pumping DNA and assemble the mature virus has been a longstanding conundrum in the field. We determined the structure of the portal, the proteins that connect it to the tail, and portal vertex in the hyperthermophilic phage Oshimavirus using cryo-Electron Microscopy (cryo-EM). We find highly intertwined loop structures, like in a wicker basket, stabilizing the portal vertex against high temperatures. Moreover, we observe that the portal protrudes from the capsid in mature virions. We propose that portal is repositioned by packaged DNA, forming a pressure-sensitive switch that terminates genome packaging and triggers tail attachment in headful phages.

microbiology↗

bioRxiv2025

An antisense RNA regulates production of DnaA and affects sporulation in Bacillus subtilis

DnaA is the replication initiator and a transcription factor in virtually all bacteria. Although the synthesis and activity of DnaA are highly regulated, the mechanisms of regulation vary between organisms. We found that production of DnaA in Bacillus subtilis is regulated by an antisense RNA that overlaps with the 5 untranslated region upstream of the dnaA open reading frame. We initially observed this RNA in in vitro transcription experiments and found that its production was inhibited by DnaA. This RNA, now called ArrA for antisense RNA repressor of dnaA, is made in vivo. We identified the arrA promoter and made a mutation that greatly reduced (or eliminated) production of ArrA RNA in vitro and in vivo. In vivo, this arrA promoter mutation caused an increase in the amount of mRNA and protein from dnaA and dnaN, indicating that arrA expression normally inhibits expression of the dnaA-dnaN operon. The arrA mutation also caused a delay in sporulation that was alleviated by loss of sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in some members of the Bacillus genus, indicating that arrA has evolved in at least some endospore-forming bacteria to modulate production of DnaA and enable timely and robust sporulation. Author summaryDnaA is the highly conserved replication initiator and transcription factor found in virtually all bacteria. The synthesis and activity of DnaA are highly regulated, and different types of bacteria use different mechanisms to control this key protein. We found that DnaA production in Bacillus subtilis is inhibited by an antisense RNA that overlaps with the 5 untranslated region of the dnaA mRNA. In the absence of this antisense RNA, there was an increase in the amount of dnaA mRNA and protein. There was also a delay in sporulation that depends on sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in several members of the Bacillus genus, indicating that it has evolved to temper production DnaA and enable robust sporulation in these species.

microbiology↗

bioRxiv2023

Differences in clamp loader mechanism between bacteria and eukaryotes

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp, and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader Replication Factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the E. coli clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

biochemistry↗