The effect of ppGpp and DksA on NAD+ capped RNA species
Date
2023
Authors
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Publisher
University of Delaware
Abstract
5´ end ribonucleic acid caps play an important role in both regulation of gene expression and half-life of RNAs. Canonically, eukaryotes utilize the evolutionarily conserved methyl-7-guanosine (m7G) cap for mRNA nuclear export and to protect RNA from exonucleolytic degradation from 5´-exonucleases1. However, novel 5´ cap structures have recently been discovered and consist of mostly metabolites, usually with an ATP moiety (NAD+, NADH, CoA, and most recently, UDP-GlyNAc). The discovery of the non-canonical initiating nucleotide (NCIN) 5´- RNA caps consisting of metabolites such as were discovered by analyzing the cleaved 5´ end products with liquid chromatography mass spectrometry (LC/MS) and has subsequently opened a new area of transcriptomic research2. Although the presence of metabolite caps is surprising, these caps have now been observed in all domains of life including archaea and viruses; however, their global biological function remains elusive. Here, we explored the effect of a stringent response in Escherichia coli on the levels of NCIN NAD+ capped RNA species. Currently, it is known that during the bacterial stringent response, high concentrations of the second messenger alarmone molecule guanosine pentaphosphate (ppGpp) are observed and is known to inhibit transcription with the E. coli transcription factor DksA, upregulating and downregulating transcription of specific genes to increase survivability3. I also sought to modify the current “CapZyme-seq” method to be used with Oxford Nanopore Sequencing for the purpose of detecting and quantifying NCIN caps in vivo. By quantifying transcription levels of RNA E. coli promoters RNA1 and rrnB P1, we were able to determine that levels of RNAI NAD+-capped RNA species transcribed during the stringent response both in vivo and in vitro were not affected by varying concentrations of ppGpp and DksA.
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Keywords
DksA, Gene expression, RNA capping, RNA polymerase, Stringent response