Modulation of RNase E Activity by Alternative RNA Binding Sitesopen access
- Authors
- Kim, Daeyoung; Song, Saemee; Lee, Minho; Go, Hayoung; Shin, Eunkyoung; Yeom, Ji-Hyun; Ha, Nam-Chul; Lee, Kangseok; Kim, Yong-Hak
- Issue Date
- Mar-2014
- Publisher
- PUBLIC LIBRARY SCIENCE
- Citation
- PLOS ONE, v.9, no.3
- Journal Title
- PLOS ONE
- Volume
- 9
- Number
- 3
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/12405
- DOI
- 10.1371/journal.pone.0090610
- ISSN
- 1932-6203
- Abstract
- Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 ((24)LYDLDIESPGHEQK(37)) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5'-P-32-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 ((427)LIEEEALK(433)), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme ((65)HGFLPL*K-71). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.
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