Global profile changes in transcripts induced with a phosphate analogue: implications for gene regulation

Abstract

Previous work has shown that thiophosphate, a phosphate analogue, leads to a global shift in the distribution of cellular proteins in a variety of organisms. Thiophosphate, when added to culture media, gets incorporated into the nucleic acids of cells resulting in nuclease-resistant phosphorothioate linkages. Using Escherichia coli, as a model organism, it was found that the global changes in protein expression induced with thiophosphate could be accounted for by significant changes in the absolute transcription levels of more than 1500 genes detected via RNA seq analysis. In fact, 58% of transcripts detected in RNA seq studies using total RNA were increased an average of 44 × fold while the remaining 42% were decreased an average of 20 × fold in thiophosphate-treated cells. Furthermore, microarray results showed no correlation between the transcriptional changes observed and the known stability of the corresponding mRNAs measured. Overall, the total amount of non-ribosomal RNA accumulated in TP-treated cells was increased relative to rRNA ~ 4 × fold (1.5-6 ×). The results further indicated that metabolic changes may play a role in inducing the transcriptional profiles observed with thiophosphate. Indeed, pathway analysis of transcripts showed an increase in routes for phosphoribosyl pyrophosphate (PRPP) synthesis and related derivatives, presumably due to a reduction in RNA turnover. These results raise the possibility that the energy savings with reduced RNA turnover could lead to an increased energy charge in the cell that induces transcriptional changes leading to an increase in biosynthetic processes.

Metabolic Regulation of Cell Differentiation

“…More recently my own research has found that use of the phosphate analogue, thio-phosphate, during cell culture appears to result in an increase in the cellular energy charge [1]. This analogue reduces RNA turnover by creating phosphorothioate linkages in RNA that inhibit cellular nucleases, which then allows the accumulation of significant amounts of mRNA and other non-ribosomal RNA in the cell.…”

“…In E. coli dramatic morphological changes are also observed using thio-phosphate instead of phosphate in culture media [1]. Namely, the bacteria form extensive filaments.…”

“…Current research raises the question of whether metabolic activity is a cause or a consequence of cell differentiation [1][2]. Clearly cell differentiation results from cell specialization and the synthesis of specific macromolecules such as proteins, all of which requires energy.…”

Early Consequences of Thiophosphate Substitution in Escherichia coli: Possible Epigenetic and Energetic Factors

“…Thiophosphate, a phosphate analogue, can be used to enhance cellular biosynthesis and thereby serve as a potential archetype for cell differentiation 1,2 . The analogue likely induces these changes by increasing the cellular energy charge or state in simple cells like bacteria.…”

“…Thiophosphate reduces RNA turnover by creating phosphorothioate linkages in RNA that inhibit cellular nucleases. The energy savings from reduced RNA turnover in Escherichia coli (E. coli) appears to enable an enhanced biosynthetic profile the result of transcriptional changes rather than differential mRNA stabilization 1,2 . Previous studies demonstrate that biosynthetic activity at later stages of growth show enhancements in the transcription of genes for RNA processing enzymes, ribosomal proteins, tRNA charging enzymes, translational proteins, as well as outer membrane, plasma membrane, and periplasmic proteins 1 .…”