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Abstract
Bacteriophage T4 of Escherichia coli is one of the most studied phages. Research into it has led to numerous contributions to phage biology and biochemistry. Coding about 300 gene products, this double-stranded DNA virus is the best-understood model in phage study and modern genomics and proteomics. Ranging from viral RNA polymerase, commonly found in phages, to thymidylate synthase, whose mRNA requires eukaryotic-like self-splicing, its gene products provide a pool of fine examples for phage research. However, there are still up to 130 gene products that remain poorly characterized despite being one of the most-studied model phages. With the recent advancement of cryo-electron microscopy, we have a glimpse of the virion and the structural proteins that present in the final assembly. Unfortunately, proteins participating in other stages of phage development are absent. Here, we report our systemic analysis on 22 of these structurally uncharacterized proteins, of which none has a known homologous structure due to the low sequence homology to published structures and does not belong to the category of viral structural protein. Using NMR spectroscopy and cryo-EM, we provided a set of preliminary structural information for some of these proteins including NMR backbone assignment for Cef. Our findings pave the way for structural determination for the phage proteins, whose sequences are mainly conserved among phages. While this work provides the foundation for structural determinations of proteins like Gp57B, Cef, Y04L, and Mrh, other in vitro studies would also benefit from the high yield expression of these proteins.
Highlights
With an estimated population of 1031, the bacteriophage—a type of virus preying on a bacterium— is the most abundant organism in the Earth’s biosphere (Clokie et al, 2011)
The primary selection criterion for our proteins of interest is the absence of published homologous structure due to low sequence homology
We filtered out small peptides with fewer than 50 amino acids in sequence, membrane proteins that may require bespoke protocols, and viral structural proteins that appear in the final virion assembly
Summary
With an estimated population of 1031, the bacteriophage—a type of virus preying on a bacterium— is the most abundant organism in the Earth’s biosphere (Clokie et al, 2011). It is believed that every bacterial strain hosts at least one type of phage, making the phage the most diversified organism (Keen, 2015). With such complexity, phage research has been focused on a few model phages. Bacteriophage T4 that infect the most-studied bacteria like Escherichia coli and Bacillus subtilis (Miller et al, 2003). Escherichia virus T4 is one such example upon which studies have contributed to various aspects of viral biology since its discovery in the 1940s (Gamkrelidze and Dabrowska, 2014; Taj et al, 2014). Some of the most significant discoveries in modern biology were aided by T4 phages, including the recognition of nucleic acids as genetic material, the demonstration that genetic codons are triplets, the discovery of mRNA, DNA restriction and modification, and self-splicing of intron/exon arrangements in prokaryotes (Kutter et al, 1995; Miller et al, 2003)
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