PMID:8632981
| Citation |
Miczak, A, Kaberdin, VR, Wei, CL and Lin-Chao, S (1996) Proteins associated with RNase E in a multicomponent ribonucleolytic complex. Proc. Natl. Acad. Sci. U.S.A. 93:3865-9 |
|---|---|
| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Animals; Base Sequence; Chaperonin 60/chemistry; DNA Primers; Endoribonucleases/biosynthesis; Endoribonucleases/chemistry; Endoribonucleases/isolation & purification; Epitopes; Escherichia coli/enzymology; Escherichia coli Proteins; HSP70 Heat-Shock Proteins/chemistry; Molecular Sequence Data; Myocardium/enzymology; Peptides/chemistry; Peptides/isolation & purification; Phosphopyruvate Hydratase/chemistry; Plasmids; Point Mutation; Polymerase Chain Reaction; Polyribonucleotide Nucleotidyltransferase/biosynthesis; Polyribonucleotide Nucleotidyltransferase/isolation & purification; RNA Helicases; RNA Nucleotidyltransferases/chemistry; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/isolation & purification; Restriction Mapping; Sequence Homology, Amino Acid |
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