Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis

Abstract

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

Estimates of RelSeq, Mesh1, and SAHMex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

“…Similarly, currently, a rigorous analysis of complex nucleotide mixtures in cell extracts appears to require HPLC coupled to mass spectrometry or recently reported capillary electrophoresis, capable of detecting all four (p)ppNpp's in one run (Haas et al, 2020). Still, determining kinetic constants with these methods would be very time consuming, as each reaction for a given nucleotide concentration would have to be stopped at several time points, and then each would have to be processed separately.…”

The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions

“…This could include RpoS, which is a major transcriptional regulator of the stress response (Schellhorn, 2020) and a potential MazF target (Sauert et al, 2016). YchF could also influence the synthesis of stress-signaling molecules, like (p)ppGpp (Haas et al, 2020;Steinchen et al, 2020) or chemical chaperones, like polyphosphate (Dahl et al, 2015), which are crucial determinants of the bacterial stress response. These possibilities are currently under investigation.…”

The Dynamic SecYEG Translocon

“…These genes are significantly down-regulated during stationary phase or when cells encounter stress conditions ( Coenye and Vandamme, 2005 ; Ikegami et al, 2005 ; Starosta et al, 2014 ). The spc operon is under control of the rplN promotor and binding of RNA-polymerase is inhibited when cells enter stationary phase by the transcription factor DksA and the alarmone ppGpp, a hyper-phosphorylated guanosine derivative ( Lemke et al, 2011 ; Haas et al, 2020 ). Thus, secY expression is obviously disconnected from the regulation of the other genes within the spc operon, probably by the presence of an internal promotor.…”

“…40 μM during exponential phase up to approx. 1 mM at the transition into stationary phase or upon amino acid starvation ( Varik et al, 2017 ; Haas et al, 2020 ; Steinchen et al, 2020 ). Cellular re-programming is induced by two mechanisms: allosteric regulation of target proteins, like RNA polymerase, which leads to reduced expression of the spc -operon ( Liang et al, 1999 ; Steinchen et al, 2020 ), and competitive inhibition of GTP-binding proteins, like the ribosome assembly factor ObgE ( Sato et al, 2005 ; Persky et al, 2009 ; Feng et al, 2014 ), the initiation factor IF2 ( Diez et al, 2020 ) or elongation factor EF-G ( Mitkevich et al, 2010 ; Steinchen et al, 2020 ).…”

“…The spc operon is under Although not yet experimentally shown, it appears likely that increasing (p)ppGpp concentrations also inhibit the two GTPases SRP and FtsY, which would fine-tune the protein targeting machinery to the reduced translation rates. control of the rplN promotor and binding of RNA-polymerase is inhibited when cells enter stationary phase by the transcription factor DksA and the alarmone ppGpp, a hyper-phosphorylated guanosine derivative (Lemke et al, 2011;Haas et al, 2020). Thus, secY expression is obviously disconnected from the regulation of the other genes within the spc operon, probably by the presence of an internal promotor.…”