Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

Abstract

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)-encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild-type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster-specific dpgA mRNA were stabilized during the idiophase in the wild-type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse-labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.