Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli
Article first published online: 19 FEB 2013
© 2013 The Authors. MicrobiologyOpen published by Blackwell Publishing Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 2, Issue 2, pages 293–307, April 2013
How to Cite
Di Pietro, F., Brandi, A., Dzeladini, N., Fabbretti, A., Carzaniga, T., Piersimoni, L., Pon, C. L. and Giuliodori, A. M. (2013), Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli. MicrobiologyOpen, 2: 293–307. doi: 10.1002/mbo3.68
- Issue published online: 8 APR 2013
- Article first published online: 19 FEB 2013
- Manuscript Accepted: 28 DEC 2012
- Manuscript Revised: 18 DEC 2012
- Manuscript Received: 11 OCT 2012
- MIUR. Grant Number: PRIN 2007
Figure S1. Assessment of MRE600 ΔyfiA mutation. (A) PCR of yfiA locus. Lane 1: 100 bp DNA ladder (Fermentas, Thermo Fisher Scientific, Inc., Waltham, MA); lane 2: amplification of the Escherichia coli wt yfiA locus; lane 3: amplification of the yfiA locus after recombination with a kanamycin cassette (yfiA::kan); lane 4: amplification of the yfiA locus after kanamycin cassette excision (ΔyfiA). (B) Two-dimensional gel electrophoresis analysis of 70S ribosomal proteins isolated from E. coli MRE600 wt (left) and ΔyfiA cells (right) grown at 37°C (upper panels) or subjected to 60 min cold shock (lower panels). The spot corresponding to protein PY is circled. Further details are given in 'Experimental Procedures'.
Figure S2. In vivo protein expression after cold shock. Synthesis of the proteins in MRE600 wt (gray lines) or MRE600 ΔyfiA (black lines) was followed by giving pulses of [35S]-Promix, as described in 'Experimental Procedures', immediately before (time 0) or at the times following cold shock indicated in the graphs. After chasing with an excess of unlabeled Met and Cys, the samples were processed for the electrophoretic separation at 7%, 10%, and 15% acrylamide concentrations and for the determination of the radioactivity using a Molecular Imager (Bio-Rad GS 250). The peaks in the graphs correspond to the intensity, expressed as “arbitrary units,” of the same point of each band present in the lanes of the various gels. The black and gray arrows indicate the pick corresponding to CspA and another unidentified cold-shock protein, respectively.
Figure S3. (A) Concentration dependence of kapp2 from the binding of PY to the 30S subunit (■) or the 50S subunit (●). The y-axis intercept gives the value of the backward rate constant koff, while the slope of the lines corresponds to the forward rate constant kon of the reactions. Standard deviations were calculated from at least 10 different time courses. (B) Time courses of PY dissociation from 30S subunit (gray) or 50S subunit (black) monitored by PY_Alexa555 fluorescent change; 0.6 μmol/L of PY_Alexa555 and 0.2 μmol/L of 30S or 50S subunits were preincubated at 15°C and then rapidly mixed with the reaction buffer. Further details are given in the text and in 'Experimental Procedures'.
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