Bacteriophages are highly abundant viruses of bacteria. The major role of phages in shaping bacterial communities and their emerging medical potential as antibacterial agents has triggered a rebirth of phage research. To understand the molecular mechanisms by which phages hijack their host, omics technologies can provide novel insights into the organization of transcriptional and translational events occurring during the infection process. In this study, we apply transcriptomics and proteomics to characterize the temporal patterns of transcription and protein synthesis during the T4 phage infection of E. coli. We investigated the stability of E. coli-originated transcripts and proteins in the course of infection, identifying the degradation of E. coli transcripts and the preservation of the host proteome. Moreover, the correlation between the phage transcriptome and proteome reveals specific T4 phage mRNAs and proteins that are temporally decoupled, suggesting post-transcriptional and translational regulation mechanisms. This study provides the first comprehensive insights into the molecular takeover of E. coli by bacteriophage T4. This data set represents a valuable resource for future studies seeking to study molecular and regulatory events during infection. We created a user-friendly online tool, POTATO4, which is available to the scientific community and allows access to gene expression patterns for E. coli and T4 genes.
Enantioselective methylation is a challenging task in organic chemistry, yet often desirable in drug discovery and optimization. S-Adenosyl methionine (SAM)-dependent methyltransferases (MTases) offer a selective alternative to chemical synthesis and an abundance of potential scaffolds. The crystal structure of C3-indole MTase PsmD from Streptomyces griseofuscus, involved in the biosynthesis of the acetylcholinesterase inhibitor physostigmine, was determined via X-ray crystallography. The amino acid residues essential for catalysis were identified by site-directed mutagenesis, and a mechanism of action was proposed. Furthermore, a PsmD ortholog was identified and characterized. The variant catalyzed enantioselective C-methylation over a broad substrate scope while displaying increased stability. Using this enzyme, preparative-scale enzymatic methylation was performed in cell-free extracts in combination with an SAM recycling system, eliminating the need for cofactor supplementation.
The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5′ modification of cellular RNAs3–5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD–RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an ‘RNAylation’ reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD–RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host’s translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.
All domains of life utilize a diverse set of modified ribonucleotides that can impact the sequence, structure, function, stability, and the fate of RNAs, as well as their interactions with other molecules. Today, more than 160 different RNA modifications are known that decorate the RNA at the 5′‐terminus or internal RNA positions. The boost of next‐generation sequencing technologies sets the foundation to identify and study the functional role of RNA modifications. The recent advances in the field of RNA modifications reveal a novel regulatory layer between RNA modifications and proteins, which is central to developing a novel concept called “epitranscriptomics.” The majority of RNA modifications studies focus on the eukaryotic epitranscriptome. In contrast, RNA modifications in prokaryotes are poorly characterized. This review outlines the current knowledge of the prokaryotic epitranscriptome focusing on mRNA modifications. Here, it is described that several internal and 5′‐terminal RNA modifications either present or likely present in prokaryotic mRNA. Thereby, the individual techniques to identify these epitranscriptomic modifications, their writers, readers and erasers, and their proposed functions are explored. Besides that, still unanswered questions in the field of prokaryotic epitranscriptomics are pointed out, and its future perspectives in the dawn of next‐generation sequencing technologies are outlined.
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