The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21 1-552 (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21 1-552 trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21 1-552 trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21 1-552 are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.

Purpose: Treacher Collins syndrome (TCS) is a rare autosomal dominant mandibulofacial dysostosis, with a prevalence of 0.2-1/ 10,000. Features include bilateral and symmetrical malar and mandibular hypoplasia and facial abnormalities due to abnormal neural crest cell (NCC) migration and differentiation. To date, three genes have been identified: TCOF1, POLR1C, and POLR1D. Despite a large number of patients with a molecular diagnosis, some remain without a known genetic anomaly. Methods: We performed exome sequencing for four individuals with TCS but who were negative for pathogenic variants in the known causative genes. The effect of the pathogenic variants was investigated in zebrafish. Results: We identified three novel pathogenic variants in POLR1B. Knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype mimicking TCS that was associated with altered ribosomal gene expression, massive p53-associated cellular apoptosis in the neuroepithelium, and reduced number of NCC derivatives. Conclusion: Pathogenic variants in the RNA polymerase I subunit POLR1B might induce massive p53-dependent apoptosis in a restricted neuroepithelium area, altering NCC migration and causing cranioskeletal malformations. We identify POLR1B as a new causative gene responsible for a novel TCS syndrome (TCS4) and establish a novel experimental model in zebrafish to study POLR1B-related TCS.