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 metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in 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 rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. 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'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
Mohanty
B K
BK
Department of Genetics, University of Georgia, Athens, GA 30602, USA.
Kushner
S R
SR
D015139:Blotting, Southern; D015169:Colony Count, Microbial; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011061:Poly A; D007544:Isopropyl Thiogalactoside; D011062:Polynucleotide Adenylyltransferase; C063643:pcnB protein, E coli; D012260:Ribonucleases