Reconstitution and analysis of the multienzyme Escherichia coli RNA degradosome.
The Escherichia coli RNA degradosome is a multienzyme assembly that functions in transcript turnover and maturation of structured RNA precursors. We have developed a procedure to reconstitute the RNA degradosome from recombinant components using modular coexpression vectors. The reconstituted assembly can be purified on a scale that has enabled biochemical and biophysical analyses, and we compare the properties of recombinant and cell-extracted RNA degradosomes. We present evidence that auxiliary protein components can be recruited to the 'superprotomer' core of the assembly through a dynamic equilibrium involving RNA intermediaries. We discuss the implications for the regulation of RNA degradosome function in vivo.
Worrall
Jonathan A R
JA
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Górna
Maria
M
Crump
Nicholas T
NT
Phillips
Lara G
LG
Tuck
Alex C
AC
Price
Amanda J
AJ
Bavro
Vassiliy N
VN
Luisi
Ben F
BF
D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D035001:Host Factor 1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D009097:Multienzyme Complexes; D012322:RNA Precursors; D012329:RNA, Bacterial; D011994:Recombinant Proteins; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D010751:Phosphopyruvate Hydratase