Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease Ⅱ and poly(A) polymerase

摘要

In all living cells 3′ ends of RNA are posttranscriptionally elongated or shortened by nucleotidyl transferases and ribonucleases. The detailed analysis of the rpsO mRNA of Escherichia coli presented here demonstrates that transcription terminates in vivo at two sites located seven and eight nucleotides downstream from the GC-rich hairpin of the intrinsic terminator and that primary transcripts can be shortened by RNase Ⅱ. The shortest RNA identified in the cell result from nibbling of primary transcripts. Primary transcripts and nibbled molecules can also be adenylated by poly(A) polymerase Ⅰ (PAP Ⅰ). In addition, kinetics of decay performed in vitro demonstrate that RNase Ⅱ rapidly degrades poly(A) tails longer than 7-8 As processively while it slowly nibbles shorter tails and non adenylated RNAs distributively. Comparison of the kinetics of nibbling of oligoadenylated rpsO mRNA in vivo and in vitro lead us to conclude that the rates of shortening and elongation of the oligo(A) tails detected in vivo are very slow: about 0.5-7 nucleotides per min. We finally speculate that the slowness of oligo(A) synthesis may explain why polyadenylation does not affect the stability of mRNAs whose degradation is controlled by RNase E.