Regulation of RNA degradation by RNase

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When cystathionine gamma-synthase (CGS) mRNA of Arabidopsis is translated in vitro in the presence of S-adenosylmethionine (SAM), translation arrest is induced at Ser-94, which then induces CGS mRNA degradation. mRNA that carries the first exon coding region of CGS was prepared by in vitro translation and was used to program in vitro translation of wheat germ extract. Ribosome was stalled at the step of translocation and the peptidyl-tRNA, the translation intermediate, was located in the A-site. A series of mRNA degradation intermediates, whose 5' ends were separated by about 30 nucleotides from each other, were identified. Evidence was found that shows that these degradation intermediates correspond to ribosomes that were stacked behind the initial stalled ribosome. Poly (G) has been reported to inhibit RNase activity in the in vitro translation system of wheat germ extract. When Poly(G) was added to the translation reaction mixture, a 5'-fragment of CGS mRNA whose 3' end matches with one of the 5' -ends of the degradation intermediate. The result suggested that the mRNA degradation occurs by an endoribonucleolytic digestion.An Escherichia coli ribonuclease colicin E5 digests the anticodon loop of tRNAs for Trp, His, Asn and Asp. Colicin E5 preferentially digests GU dinucleotide. Structure and substrate specificity relationship of colicin E5 were studied. The substrate specificity could be explained by the structural constraints at the substrate pocket. Effects of expression of colicin D that digests tRNA(Arg) in budding yeast and HeLa cells were analyzed.