THE C-TERMINAL DISORDERED REGION OF THE RNA HELICASE DEAD IS REQUIRED FOR COLD SHOCK GROWTH IN E. COLI
Date
2025-05
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Publisher
University of Delaware
Abstract
DeaD, a protein belonging to the dead box family of RNA helicases in
Escherichia coli (E. coli), has many cellular functions. The known functions of DeaD
include its function as an RNA helicase, a role in ribosome biogenesis and enabling
growth at cold shock temperatures. DeaD expression is upregulated in cold
temperatures, which has led to it being given an alternative name: cold shock DeaD
box protein A (CsdA). We want to explore the function of DeaD during E. coli growth
under cold shock conditions. Most of the protein’s structure has been attributed to the
RNA helicase function. In addition to the RNA helicase domains, DeaD has two
intrinsically disordered regions (IDRs) and a DbpA domain. The function of these
other regions is less well researched. Using epifluorescence microscopy and growth
assays, I have been able to outline the significance of the C-terminal IDR to DeaD's
primary functions. In addition to assessing the function of DeaD as a cold shock
protein, we also want to characterize its localization within the cell.
Dead and other Deadbox helicases have been studied for their role in forming
RNA condensates. This phase-phase separation is important to cellular organization
and has been observed in both prokaryotic and eukaryotic cells. Since the C-terminal
IDR and DbpA domain have been implicated as RNA-binding domains, I wanted to
see if DeaD could form condensates without the C-terminal IDR. By fusing the DeaD
protein with a fluorescent protein, we can use microscopy to track protein localization
inside the cell. A deaD deletion strain of E. coli was transformed with tetracycline
inducible plasmids encoding either the full-length dead or the truncated form tagged
with a fluorescent protein. Full-length DeaD was able to form puncta at both preferred
temperature and cold shock, while the truncation was not entirely successful. Based on
our results, we have concluded that functioning DeaD is necessary for cells to survive
cold shock conditions and for this localization to occur.