Temporal gene expression and regulation in T4 phage
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Levine_mlevine1_PhD_MICRO_2024_Thesis.pdf
Description
Thesis PDF
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3.5 MB
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Adobe PDF
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aa35cf18b31385a385eea7308878e26c
Author(s)
Levine, Mandara Alexis
Advisor(s)
Laub, Michael T.
Li, Gene-Wei
Date Issued
February 2024
Publisher
Massachusetts Institute of Technology
Abstract
As the most abundant biological entity in the biosphere, bacteriophages play a critical role in shaping the microbial diversity, and thus overall ecosystem health. They are also essential tools in molecular biology, shedding light on fundamental biological concepts. T4 phage, with its complex lifecycle and genetic content, has been instrumental in many such discoveries. However, many questions regarding gene regulation in T4 phage remain unanswered. In this study, we employ end-enriched RNA-seq (Rend-seq) and ribosome profiling to examine T4 RNA and protein synthesis throughout the course of infection, gaining new insights at the transcriptional, translational, and genomic level. At the transcriptional level, we identified transcript boundaries, novel putative promoters, and new potential cleavage sites for the T4 endoribonuclease RegB. At the translational level, we identified many instances of previously unreported changes in translational efficiency over the course of infection, indicating the presence of intricate and uncharacterized mechanisms of regulation. Collectively, transcriptional and translational controls lead to precisely tuned protein synthesis rates during infection, as exemplified by the phenomenon that components of T4 protein complexes are synthesized according to their stoichiometry— a principle that has been observed in organisms during steady-state growth. Finally, we identified and experimentally validated T4’s 290th gene, 61.-1. Though non-essential to T4 in laboratory conditions, this gene has homologs present in a number of other phage and drastically impacts E. coli growth when ectopically expressed. This study provides insights into T4 phage biology, paving the way for further exploration into molecular biology, virology, and biotechnology; our rich data set can be utilized by future studies to answer a diverse array of inquiries.
MIT Department
Massachusetts Institute of Technology. Microbiology Graduate Program
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