title: Modulation of RNase E activity by alternative RNA binding sites.
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 (24LYDLDIESPGHEQK37) 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'-32P-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 (427LIEEEALK433), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71). 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.
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Daeyoung
authors.initials: D
authors.lastname: Kim
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Saemee
authors.initials: S
authors.lastname: Song
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Minho
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea; Department of Genetics, Stanford University, Stanford, California, United States of America.
authors.forename: Hayoung
authors.initials: H
authors.lastname: Go
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Eunkyoung
authors.initials: E
authors.lastname: Shin
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Ji-Hyun
authors.initials: JH
authors.lastname: Yeom
authors.affiliation: Department of Manufacturing Pharmacy, Pusan National University, Busan, Republic of Korea.
authors.forename: Nam-Chul
authors.initials: NC
authors.lastname: Ha
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Kangseok
authors.initials: K
authors.lastname: Lee
authors.affiliation: Department of Microbiology, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea.
authors.forename: Yong-Hak
authors.initials: YH
authors.lastname: Kim
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D059367:RNA Cleavage; D012329:RNA, Bacterial / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D004722:Endoribonucleases; C022537:ribonuclease E