It has been more than 50 years since the discovery of dinucleoside polyphosphates (Np n Ns) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated Np n Ns serve as RNA caps in Escherichia coli. Np n Ns are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5′-pyrophosphohydrolase (RppH) and bis(5′-nucleosyl)-tetraphosphatase (ApaH) are able to remove the Np n N-caps from RNA. ApaH is able to cleave all Np n N-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as Np n Ns are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
Dinucleoside polyphosphates (Np n Ns) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5′ RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed Np n Ns to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of Ap n Gs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the −1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.
Calix[4]arenes bearing diaryl urea moieties with electron-withdrawing substituents on both sides can bind selected anions (AcO−, BzO−, H2PO4−) even in highly competitive solvents such as DMSO-d6.
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