Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

Abstract

The pnp gene, encoding the enzyme polynucleotide phosphorylase (PNPase), was overexpressed in the actinomycin producer Streptomyces antibioticus. Integration of pIJ8600, bearing the thiostrepton-inducible tipA promoter, and its derivatives containing pnp into the S. antibioticus chromosome dramatically increased the growth rate of the resulting strains as compared with the parent strain. Thiostrepton induction of a strain containing pJSE340, bearing pnp with a 5′-flanking region containing an endogenous promoter, led to a 2·5–3 fold increase in PNPase activity levels, compared with controls. Induction of a strain containing pJSE343, with only the pnp ORF and some 3′-flanking sequence, led to lower levels of PNPase activity and a different pattern of pnp expression compared with pJSE340. Induction of pnp from pJSE340 resulted in a decrease in the chemical half-life of bulk mRNA and a decrease in poly(A) tail length as compared to RNAs from controls. Actinomycin production decreased in strains overexpressing pnp as compared with controls but it was not possible to attribute this decrease specifically to the increase in PNPase levels. Overexpression of pnp had no effect on ppGpp levels in the relevant strains. It was observed that the 3′-tails associated with RNAs from S. antibioticus are heteropolymeric. The authors argue that those tails are synthesized by PNPase rather than by a poly(A) polymerase similar to that found in Escherichia coli and that PNPase may be the sole RNA 3′-polynucleotide polymerase in streptomycetes.

RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase

“…4 revealed bands in all three preparations that were 25-35 residues in length. This observation confirms previous results that while oligo(A) stretches of less than 18-20 residues are most commonly observed as components of the heteropolymeric 39-tails of streptomycete RNAs, longer stretches do occur at a lower frequency (Bralley & Jones, 2003). It should be noted that because the poly(A) tails of streptomycete RNAs are highly heteropolymeric (Bralley & Jones, 2002), the pattern of continuous increase in tail length observed in E. coli (lane 3) is not observed for the tails from S. coelicolor.…”

“…These cDNAs, from leuA (SCO0387), clpP (SCO3044) and a 23S rRNA, were isolated from both wildtype and mutant strains. These clones revealed tails with compositions similar to those previously isolated from genes actII-orf4 and redD (Bralley & Jones, 2002) and S. antibioticus pnp (Bralley & Jones, 2003). The fact that both RNA tail length and composition in the mutant JSE450A appear similar to that found in M145 strongly suggests that RNase PH is not involved in the synthesis of RNA 39-tails in S. coelicolor.…”

“…Most interestingly, when the nature of the 39-tails associated with S. coelicolor RNAs was examined by cDNA cloning and DNA sequencing, it was observed that those tails were heteropolymeric, containing G, C and U as well as A residues (Bralley & Jones, 2002). Heteropolymeric tails were also observed at the 39-ends of RNAs from Streptomyces antibioticus and we therefore believe that such tails are likely to be found in all streptomycetes (Bralley & Jones, 2003).…”

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…Overexpression of PNPase led to decreased antibiotic production in Streptomyces antibioticus (155). Since the expression of pnp is RNase III dependent in S. coelicolor (156), it will be interesting to investigate the connection of PNPase, RNase III, and antibiotic production.…”

Organization and Expression of the Polynucleotide Phosphorylase Gene ( pnp ) of Streptomyces : Processing of pnp Transcripts in Streptomyces antibioticus

“…More recently, it has been shown that PNPase also functions as an alternative poly(A) polymerase in E. coli, where under appropriate conditions it can either degrade RNA or synthesize poly(A) tails, also incorporating C and U residues at low frequency in wild-type cells (34). It has further been argued that PNPase serves as perhaps the sole poly(A) polymerase in streptomycetes (7,8), spinach chloroplasts (53), and Synechocystis sp. (46), synthesizing heteropolymeric rather than homopolymeric 3Ј-RNA tails in each of these systems.…”