No abstract

RNase E isolated from Escherichia coli is contained in a multicomponent ''degradosome'' complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.immunogold labeling ͉ RhlB RNA helicase ͉ RNA process ͉ RNA degradation R Nase E is an essential Escherichia coli ribonuclease that has a key role in the degradation and͞or processing of both short and long half-lived RNAs. When purified from E. coli cells, RNase E is present in a multicomponent ribonucleolytic complex (i.e., the RNA ''degradosome'') that includes polynucleotide phosphorylase (PNPase), the RhlB RNA helicase, enolase, the DnaK chaperonin protein, GroEL, and polynucleotide phosphate kinase (PPK) (1-5). Specific regions required to bind certain degradosome proteins have been identified within the C-terminal half of RNase E (6), and a functionally active minimal degradosome containing only RNase E, PNPase, and helicase has been reconstituted in vitro (7).The view that many, if not most, cellular functions are carried out in vivo by multicomponent macromolecular complexes (i.e., cellular machines) rather than by individual freely diffusable proteins has gained wide acceptance in recent years (8). Well recognized and extensively studied examples of such complexes in bacteria and higher organisms include ribosomes, replisomes, and proteasomes (9-11). However, notwithstanding the isolation of multicomponent RNase E-based complexes from E. coli (1-5, 12), there has been no direct evidence that degradosomes are present in living cells-rather than being formed in vitro as aggregates of individual proteins. The question of whether degradosomes actually exist in vivo in E. coli is especially relevant in view of evidence that truncated RNase E protein lacking the C-terminal half is sufficient for cell viability and for RNA degradation and processing in vivo in E. coli (13,14), that RNase E homologs in certain other bacteria do not contain the scaffold region that interacts with PNPase and other degradosome proteins (15), and that puri...