Proteins associated with RNase E in a multicomponent ribonucleolytic complex.
The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.
Miczak
A
A
Institute of Molecular Biology, Academia Sinica, Nankang Taipei, Taiwan, Republic of China.
Kaberdin
V R
VR
Wei
C L
CL
Lin-Chao
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018834:Chaperonin 60 / Q000737:chemistry; D017931:DNA Primers; D004722:Endoribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010751:Phosphopyruvate Hydratase / Q000737:chemistry; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011117:Polyribonucleotide Nucleotidyltransferase / Q000096:biosynthesis / Q000302:isolation & purification*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D018834:Chaperonin 60; D017931:DNA Primers; D000939:Epitopes; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D012316:RNA Nucleotidyltransferases; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; C063842:dnaK protein, E coli; D020365:RNA Helicases; D010751:Phosphopyruvate Hydratase