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RNase II is important for A‐site mRNA cleavage during ribosome pausing
Author(s) -
GarzaSánchez Fernando,
Shoji Shinichiro,
Fredrick Kurt,
Hayes Christopher S.
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2009.06813.x
Subject(s) - rnase p , biology , ribosome , polynucleotide phosphorylase , cleavage (geology) , cleavage factor , ribosomal binding site , rnase ph , a site , cleavage and polyadenylation specificity factor , exoribonuclease , exonuclease , exosome complex , microbiology and biotechnology , start codon , rnase mrp , endoribonuclease , messenger rna , biochemistry , binding site , rna , polyadenylation , gene , enzyme , purine nucleoside phosphorylase , paleontology , purine , fracture (geology) , dna polymerase
Summary In Escherichia coli , translational arrest can elicit cleavage of codons within the ribosomal A site. This A‐site mRNA cleavage is independent of RelE, and has been proposed to be an endonucleolytic activity of the ribosome. Here, we show that the 3′→5′ exonuclease RNase II plays an important role in RelE‐independent A‐site cleavage. Instead of A‐site cleavage, translational pausing in ΔRNase II cells produces transcripts that are truncated +12 and +28 nucleotides downstream of the A‐site codon. Deletions of the genes encoding polynucleotide phosphorylase (PNPase) and RNase R had little effect on A‐site cleavage. However, PNPase overexpression restored A‐site cleavage activity to ΔRNase II cells. Purified RNase II and PNPase were both unable to directly catalyse A‐site cleavage in vitro . Instead, these exonucleases degraded ribosome‐bound mRNA to positions +18 and +24 nucleotides downstream of the ribosomal A site respectively. Finally, a stable structural barrier to exoribonuclease activity inhibited A‐site cleavage when introduced immediately downstream of paused ribosomes. These results demonstrate that 3′→5′ exonuclease activity is an important prerequisite for efficient A‐site cleavage. We propose that RNase II degrades mRNA to the downstream border of paused ribosomes, facilitating cleavage of the A‐site codon by an unknown RNase.