Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners.
RNase E is an essential enzyme that catalyses RNA processing. Microdomains which mediate interactions between RNase E and other members of the degradosome have been defined. To further elucidate the role of these microdomains in molecular interactions, we studied RNase E from Vibrio angustum S14. Protein sequence analysis revealed that its C-terminal half is less conserved and structured than its N-terminal half. Within this structural disorder, however, exist five small regions of predicted structural propensity. Four are similar to interaction-mediating microdomains identified in other RNase E proteins; the fifth did not correspond to any known functional motif. The function of the V. angustum S14 enolase-binding microdomain was confirmed using bacterial two-hybrid analysis, demonstrating the conserved function of this microdomain for the first time in a species other than Escherichia coli. Further, PNPase in V. angustum S14 was shown to interact with the last 80 amino acids of the C-terminal region of RNase E. This raises the possibility that PNPase interacts with the small ordered region at residues 1026-1041. The role of RNase E as a hub protein and the implications of microdomain-mediated interactions in relation to specificity and function are discussed.
Systems Biology Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
Melissa A
MA
Erce
Jason K K
JK
Low
Paul E
PE
March
Marc R
MR
Wilkins
Kathy M
KM
Takayama
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009097:Multienzyme Complexes / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D020365:RNA Helicases / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014733:Vibrio / Q000201:enzymology
D009097:Multienzyme Complexes; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D020365:RNA Helicases; D010751:Phosphopyruvate Hydratase