Figure S1: Cellular organization of RhlB-His and PNPase
Cellular organization of RhlB-His (Panels A-B) and PNPase (Panels C-D) by immunofluorescence microscopy using purified anti-RhlB or anti-PNPase antibodies respectively (4). Panel A: showing helical organization of RhlB-His in AT36/ pAT66 [∆rhlB/Plac-rhlB::(His)6] cells. Panel B: showing helical organization of RhlB-His in AT40/ pAT66 [∆rhlB, rne1-417/Plac-rhlB::(His)6] cells. Cells with rne1-417truncation lacked the RNaseE helical structures and grew as mixed cell population with varying cell length including filamentous cells (4). Panel C: showing diffusely distributed PNPase in AT40 [∆rhlB, rne1-417] cells that lacked both the RhlB and RNaseE coiled structures. Panel D: showing helical organization of PNPase in AT40/ pAT66 [∆rhlB, rne1-417/Plac-rhlB::(His)6] cells that lacked the RNaseE structures due to rne1-417mutation. Panel E: quantitative immunoblot to determine the cellular concentration of RhlB. MC1000 cells (1) were grown at 37 C in LB medium to OD600 0.8. An SDS cell extract was prepared from 4 ml cultures as previously described (3). Cell extract (1 µl (lane 5), 2.5 µl (lane 6) and 5 µl (lane 7)) and purified RhlB-His (3 ng (lane 1), 7.5 ng (lane 2), 15 ng (lane 3), and 30 ng (lane 4)) were loaded on the same gel and electrophoresed. Western blot analysis was performed using affinity-purified anti-RhlB (4). RhlB bands were quantitated by densitometry using the ImageQuant program (Molecular Dynamics). Number of RhlB molecules per cell was estimated assuming that the volume of an E. coli cell is 1 fL (2).
Table S1:Initial rates of RhlB polymerization
Nucleotides / Polymerization rate (a. u. /min)aATP / 0.9 0.09
AMP-PNP / 1.2 0.17
ATP--S / 1.0 0.16
ADP / 0.3 0.04
a/ Initial polymerization rates of RhlB were determined by calculating the slope of the linear best-fit plot between time-points 0 and 2.5 min.
References
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2.Sundararaj, S., A. Guo, B. Habibi-Nazhad, M. Rouani, P. Stothard, M. Ellison, and D. S. Wishart. 2004. The CyberCell Database (CCDB): a comprehensive, self-updating, relational database to coordinate and facilitate in silico modeling of Escherichia coli. Nucleic Acids Res 32:D293-5.
3.Taghbalout, A., and L. Rothfield. 2008. RNaseE and RNA helicase B play central roles in the cytoskeletal organization of the RNA degradosome. J. Biol. Chem. 283:13850-5.
4.Taghbalout, A., and L. Rothfield. 2007. RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton. Proc Natl Acad Sci U S A 104:1667-72.
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