Localization of Escherichia coli poly(A) polymerase I in cellular membrane

Poly(A) polymerase I (PAP I), the pcnB gene product, is the main enzyme responsible for RNA polyadenylation in Escherichia coli. Polyadenylated RNA molecules are rapidly degraded by a multiprotein complex called RNA degradosome. Here we demonstrate that apart from its presence in cytosol, PAP I is also localized in cellular membrane. Although this observation might appear surprising, it was demonstrated recently by others that E. coli RNA degradosome is also associated with the cytoplasmic membrane. Moreover, we show that development of single-stranded RNA bacteriophages MS2 and Qbeta, but not that of single-stranded DNA bacteriophage M13, is more efficient in the pcnB mutant relative to an otherwise isogenic pcnB(+) host.     

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).     

Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.     

Deregulation of poly(A) polymerase I in Escherichia coli inhibits protein synthesis and leads to cell death

Polyadenylation plays important roles in RNA metabolism in both prokaryotes and eukaryotes. Surprisingly, deregulation of polyadenylation by poly(A) polymerase I (PAP I) in Escherichia coli leads to toxicity and cell death. We show here that mature tRNAs, which are normally not substrates for PAP I in wild-type cells, are rapidly polyadenylated as PAP I levels increase, leading to dramatic reductions in the fraction of aminoacylated tRNAs, cessation of protein synthesis and cell death. The toxicity associated with PAP I is exacerbated by the absence of either RNase T and/or RNase PH, the two major 3′ → 5′ exonucleases involved in the final step of tRNA 3′-end maturation, confirming their role in the regulation of tRNA polyadenylation. Furthermore, our data demonstrate that regulation of PAP I is critical not for preventing the decay of mRNAs, but rather for maintaining normal levels of functional tRNAs and protein synthesis in E. coli, a function for polyadenylation that has not been observed previously in any organism.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.