title: The catalytic domain of RNase E shows inherent 3' to 5' directionality in cleavage site selection.
abstract: RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2'-O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5'-monophosphate RNA termini but has an inherent mode of cleavage in the 3' to 5' direction. Target sequences made uncleavable by the introduction of 2'-O-methyl-modified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5' to, but not 3' to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3' to 5' "scanning" mechanism and imply that anchoring of the enzyme to the 5'-monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, CA 94305USA-5120, USA.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Feng
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Vickers
authors.forename: Stanley N
authors.initials: SN
authors.lastname: Cohen
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D013379:Substrate Specificity
chemical_list: D009843:Oligoribonucleotides; D004722:Endoribonucleases; C022537:ribonuclease E