title: Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding.
abstract: Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5'-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. However, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart: two substrate-bound tryptic peptides, (65) HGFLPLK (71)--which includes amino acids previously implicated in substrate binding and catalysis--and (24) LYDLDIESPGHEQK (37)--which includes the Q36 locus-were identified in wild-type enzyme complexes. Only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.
authors.affiliation: School of Biological Sciences, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Hayoung
authors.initials: H
authors.lastname: Go
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Moore
authors.forename: Minho
authors.initials: M
authors.lastname: Lee
authors.forename: Eunkyoung
authors.initials: E
authors.lastname: Shin
authors.forename: Che Ok
authors.initials: CO
authors.lastname: Jeon
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Cha
authors.forename: Seung Hyun
authors.initials: SH
authors.lastname: Han
authors.forename: Su-Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Younghoon
authors.initials: Y
authors.lastname: Lee
authors.forename: Nam-Chul
authors.initials: NC
authors.lastname: Ha
authors.forename: Yong-Hak
authors.initials: YH
authors.lastname: Kim
authors.forename: Stanley N
authors.initials: SN
authors.lastname: Cohen
authors.forename: Kangseok
authors.initials: K
authors.lastname: Lee
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012313:RNA / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D029968:Escherichia coli Proteins; D012313:RNA; D004722:Endoribonucleases; C022537:ribonuclease E