Host Takeover by Bacteriophage T4

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

It is well-established that in the phage biosphere a large proportion of genes do not have homologs outside of phage. Consequently, these genes represent a unique repository of genetic information of unknown functions and mechanisms. For bacteriophage T4, approximately one third of its genome is expressed from early genes within the first minute of infection. Many of these early genes are uncharacterized and are nonessential under normal laboratory conditions, but they are thought to be involved in phage takeover of the Escherichia coli host cell. We have investigated 3 such early T4 genes (goF, motB.1, and frd.2) because our deep BLAST analyses predicted that each one contains an Lsm fold. This motif, which is found throughout biology, is frequently involved in interactions with RNA. For example, the major bacterial RNA chaperone Hfq is an Lsm fold protein, responsible for post-transcriptional regulation through its interactions with mRNA and mediation of sRNA/mRNA associations. Although the functions of the T4 genes are unknown, a mutant GoF(D25Y) has been shown to increase the levels of both the T4 gene 41 RNA and 41 protein under certain conditions, suggesting that it might be involved with RNA. It was first postulated that GoF(D25Y) did this by being a T4 anti-terminator of transcription. However, other work suggested that GoF(D25Y) worked post-transcriptionally. Understanding the functions of GoF, MotB.1, and Frd.2 has been hampered by the lack of homologs outside the phage world in the protein sequence database. Thus, the standard approach of finding a sequence homolog or motif to direct one toward possible functions has not been applicable. Because of this, we leveraged the most recent protein structure prediction program, AlphaFold3, together with biochemical assays to investigate possible functions for the proteins. AlphaFold3 confirmed the presence of Lsm-like motifs in each of the 3 T4 proteins and suggested the structural location of the D to Y substitution within the mutant GoF. To investigate the function of GoF(D25Y), we used a tandem fluorescence translational reporter assay with a reporter mCherry gene. In this system, wild type goF, goF(D25Y), or motB.1 were present on a plasmid and placed downstream of the arabinose-inducible promoter PBAD. In the chromosome, a strong constitutive promoter (Pcon) was located upstream of 2 reporter genes, mCherry followed by gfp. gfp had its own ribosome binding site (RBS) while various RBS-containing sequences were placed upstream of mCherry for testing. Because previous work had shown a significant effect of GoF(D25Y) on T4 gene 41 expression, we used the 358 bp region upstream of T4 41 (41 5’UR) as well as just the 20 bp immediately upstream of 41 (41 5’UR 20 nt), since these regions contain the natural sequence present in the T4 genome. However, because we knew that GoF(D25Y) can affect the levels of several proteins, we also tested other general RBSs. To quantify our results, we measured the growth of the cells as monitored by cell density and the levels of mCherry and GFP fluorescence. We then normalized the fluorescence relative to the cell density and determined the ratio of the fluorescence observed with the induced plasmid relative to the uninduced plasmid. Thus, if production of the T4 protein increased the level of mCherry fluorescence, without a corresponding change in the level of GFP fluorescence, we could conclude that mCherry expression is altered post-transcriptionally. This could arise from a change in the stability of the RNA and/or by a change in translation. Using this assay, we found that the presence of GoF(D25Y) resulted in a significant increase in both the level of the 41 5’UR-mCherry and the 41 5’UR 20 nt-mCherry fluorescence and a significant increase in the level of their RNAs. Thus, GoF(D25Y) works post-transcriptionally by increasing the half-life of the RNA, indicating that GoF(D25Y) either protects the RNA by directly binding to it or it works indirectly by interfering with nucleases and/or by increasing translation, which can also increase RNA stability. Interestingly, we did not observe the same effect using either WT GoF or MotB.1. This suggests that the D25Y substitution imparts a significant change in GoF activity. Our AlphaFold3 predicted structures of WT GoF and GoF(D25Y) indicated that the D to Y substitution introduces a dramatic conformational change, which generates a positive electrostatic potential on the protein surface within the Lsm-like fold. Such a change would be consistent with a gain of function mutation that increases the ability of the LSm-like fold to bind RNA. Understanding how gain of function happens is of utmost importance, as such mutations are involved in dynamic biological processes, such as evolution and tumor progression. Recent work indicates that these mutations need to be considered in the context of structure, not sequence. However, previous studies have shown that predicting the structural and functional effects of a point mutation are challenging, and only a limited number of wild type vs. mutant proteins have been evaluated to assess how well protein structure prediction programs perform. Consequently, our studies with GoF and GoF(D25Y) provide a model system for investigating gain-of-function mutants and reveals the power of AlphaFold3 to find unexpected structure/function relationships among uncharacterized proteins.