Poster abstracts
Poster number 54 submitted by Aishwarya Ghosh
Bacterial translation initiation factor IF-2’s N-terminal IDR promotes cold-induced phase separation
Aishwarya Ghosh (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Vidhyadhar Nandana (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Kaveendya Mallikaarachchi (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Jared M. Schrader (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI)
Abstract:
Bacterial cells, face challenges in organizing biochemical pathways because they generally lack membrane-bound organelles and have limited space. Biomolecular condensates (BMCs) are non-membrane bound organelles often assembled by phase-separation and offer a potential solution to organize their biochemical reactions in bacteria. BMCs composed of the machinery for DNA replication (1), transcription (2), and mRNA decay (3) have been identified. However, no BMCs for the mRNA translation machinery have been documented in bacteria. Our bioinformatic analyses of the bacterial translation machinery revealed that translation initiation factor 2 (IF-2) possesses a large intrinsically disordered region (IDR), suggesting it likely forms a BMC. To investigate the hypothesis that bacterial IF-2 can phase separate, we studied IF-2 from Escherichia coli and Caulobacter crescentus using both in vivo imaging and in vitro reconstitution approaches. Immunofluorescence results revealed that IF-2 localizes in foci within the cells of both species. We investigated the ability of IF-2 to phase-separate by purifying the IF-2 protein and conducting in vitro droplet formation assays. Our results demonstrate that IF-2 can phase separate in an RNA-dependent manner and that deleting the IDR strongly reduces phase separation. The deletion of the IDR in E.coli IF-2 was previously shown to yield a cold sensitive phenotype (4), but the source of cold sensitivity was not established. To investigate the role of IF-2 phase separation in E. coli cold sensitivity, we examined how temperature affects IF-2 phase separation. Our in vitro reconstitution experiments demonstrated that E. coli IF-2 phase separation is enhanced at lower temperatures and the cold-enhancement requires the IDR, suggesting that reduced phase separation of the IDR deletion may be the cause of cold sensitive growth. In conclusion, IF-2 phase separation provides the first evidence that bacteria possess translation machinery BMCs. Given the essential nature of central dogma processes in bacteria, IF-2 BMCs represent a potential new target for next-generation antibiotics against pathogenic E.coli that survive in refrigerated food.
References:
1. Harami, Gábor M., et al. "Phase separation by ssDNA binding protein controlled via protein− protein and protein− DNA interactions." Proceedings of the National Academy of Sciences 117.42 (2020): 26206-26217.
2. Ladouceur, Anne-Marie, et al. "Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation." Proceedings of the National Academy of Sciences 117.31 (2020): 18540-18549.
3. Al-Husini, Nadra, et al. "α-Proteobacterial RNA degradosomes assemble liquid-liquid phase-separated RNP bodies." Molecular cell 71.6 (2018): 1027-1039.
4. Brandi, Anna, et al. "Translation initiation factor IF2 contributes to ribosome assembly and maturation during cold adaptation." Nucleic Acids Research 47.9 (2019): 4652-4662.
Keywords: Phase separation, Biomolecular condensate, Translation initiation factor IF-2