RNase II is important for A-site mRNA cleavage during ribosome pausing.
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 DeltaRNase 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 DeltaRNase 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.
Garza-Sánchez
Fernando
F
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106-9610, USA.
Shoji
Shinichiro
S
Fredrick
Kurt
K
Hayes
Christopher S
CS
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D011117:Polyribonucleotide Nucleotidyltransferase / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C022535:exoribonuclease II