Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

To help understand the role of polyadenylation in Escherichia coli RNA meta bolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affect ing cell growth or viability. Increased polyadenylation led to a decrease i n the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for me (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes in volved in mRNA decay, were stabilized. mb (RNase II) and rnc (RNase III) tr anscript levels were unaffected in the presence of increased polyadenylatio n. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminu s of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an al most 20-fold variation in the level of polyadenylation among three differen t transcripts and that PAP I accounted for between 94% and 98.6% of their p oly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polym erized into poly(A) tails in a transcript-dependent manner.