RNaseE and RNA Helicase B Play Central Roles in the Cytoskeletal Organization of the RNA Degradosome

Aziz Taghbalout,Lawrence Rothfield

doi:10.1074/jbc.m709118200

Abstract

The RNA degradosome of Escherichia coli is a multiprotein complex that plays an essential role in normal RNA processing and decay. It was recently shown that the major degradosome constituents are organized in a coiled cytoskeletal-like structure that extends along the length of the cell. Here we show that the endoribonuclease E (RNaseE) and RNA helicase B (RhlB) components of the degradosome can each independently form coiled structures in the absence of the other degradosome proteins. In contrast, the cytoskeletal organization of the other degradosome proteins required the presence of the RNaseE or RhlB coiled elements. Although the RNaseE and RhlB structures were equally competent to support the helical organization of polynucleotide phosphorylase, the cytoskeletal-like organization of enolase occurred only in the presence of the RNaseE coiled structure. The results indicate that the RNA degradosome proteins are components of the bacterial cytoskeleton rather than existing as randomly distributed multiprotein complexes within the cell and suggest a model for the cellular organization of the components within the helical degradosomal structure.

Highlights

Several of the bacterial cytoskeletal elements are organized as helical filamentous structures that wind around the cell from pole to pole, associated with the inner surface of the cytoplasmic membrane
We have recently shown that the major protein components of the RNA degradosome are localized as helical cytoskeletal-like structures within the cell [5]
In this study we investigated the roles of the different degradosome proteins in the helical organization of the degradosome

Summary

EXPERIMENTAL PROCEDURES

Plasmids, Media, and Growth Condition—Escherichia coli strains were grown in Luria Bertani medium [17] to which 100 ␮g/ml ampicillin, 25 ␮g/ml kanamycin, 30 ␮g/ml chloramphenicol, or 0.4% (w/v) glucose were added when indicated. ⌬eno strains were grown in M9 medium supplemented with 0.2% tryptone/0.2% glycerol/1 mM MgSO4/0.0001% thiamine and 40 mM succinate. Plasmids, Media, and Growth Condition—Escherichia coli strains were grown in Luria Bertani medium [17] to which 100 ␮g/ml ampicillin, 25 ␮g/ml kanamycin, 30 ␮g/ml chloramphenicol, or 0.4% (w/v) glucose were added when indicated. Plasmids and strains are listed, and the details of their construction are available upon request. Hemagglutinin (HA) epitope tagging was done as previously described [20], and the associated antibiotic cassettes were eliminated, when indicated, by use of the FLP recombinase expressing plasmid pCP20 [19]. Microscopy—Yfp-labeled cells were examined by fluorescence microscopy as previously described [22]. Rabbit antisera directed against RhlB and against PNPase, kindly provided by Dr M. Immunoblotting Analysis—Quantitative immunoblotting was done on 5, 10, and 30 ␮g of protein of total cell extracts as previously described [22], using 12% SDS-PAGE

RESULTS

Reference or source

Wild type strain

DISCUSSION