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

Abstract

SummaryTo 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 halflives 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. Longterm 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 3Ј-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.

cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor

“…Two 23S rRNA clones (3134 and 3128) were polyadenylated at 3h immature precursor sites similar to those found in E. coli (Mohanty & Kushner, 1999, 2000. All the other clones showed polyadenylation deep within the coding regions (Fig.…”

“…Applying similar RT-PCR methods to those used here, Mohanty & Kushner (1999) showed in E. coli that 6\8 lpp clones were homopolymers, while two clones contained one T and C residue, respectively. A similar predominance of high A content tails has been reported for the flagellin mRNA of Bacillus subtilis and bacteriophage T7 mRNA (Cao & Sarkar, 1993, Johnson et al, 1998.…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…The data led to the model, subsequently proven, that precursor hypomodified tRNA i Met is 39 polyadenylated by Trf4 and the poly(A)-containing tRNA is degraded by the nuclear exosome (Kadaba et al 2004(Kadaba et al , 2006 (Table 1; Figure 5). Poly(A) tails on mRNA generally specify stability; however, in E. coli RNA turnover also proceeds by poly(A) addition (Mohanty and Kushner 1999). Trf4-mediated poly(A) addition is also involved in the turnover of other types of aberrant transcripts (Kadaba et al 2006).…”

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

“…PNPase is also responsible for the G, C and U residues that are found at low frequency in the poly(A) tails of RNAs from wild-type E. coli (Mohanty & Kushner, 2000b). The overall model for RNA degradation that has emerged from the studies in E. coli involves the generation of RNA 39-ends via endonucleolytic cleavage by the endoribonucleases RNase E and RNase III, polyadenylation of the 39-ends by PAP I (and under some conditions by PNPase) and degradation of the resulting RNAs exonucleolytically by PNPase (with the assistance of the DEAD box helicase RhlB), RNase II and RNase R (Cheng & Deutscher, 2005;Coburn & Mackie, 1999;Mohanty & Kushner, 1999b, 2000aO'Hara et al, 1995). This model applies especially to structured RNAs, with hairpins at their 39-ends (Carpousis et al, 1999;Régnier & Arraiano, 2000).…”