Quantifying ribosome dynamics in Escherichia coli using fluorescence.
Ribosomes are a crucial component of the physiological state of a cell. Therefore, we aimed to monitor ribosome dynamics using a fast and easy fluorescence readout. Using fluorescent-labeled ribosomal proteins, the dynamics of ribosomes during batch cultivation and during nutritional shift conditions was investigated. The fluorescence readout was compared to the cellular rRNA content determined by capillary gel electrophoresis with laser-induced fluorescence detection during exponentially accelerating and decelerating growth. We found a linear correlation between the observed fluorescence and the extracted rRNA content throughout cultivation, demonstrating the applicability of this method. Moreover, the results show that ribosome dynamics, as a result of slowing growth, are accompanied by the passive effect of dilution of preexisting ribosomes, de novo ribosome synthesis and ribosome degradation. In light of the challenging task of deciphering ribosome regulatory mechanisms, our approach of using fluorescence to follow ribosome dynamics will allow more comprehensive studies of biological systems.
Jurek
J
Failmezger
Julian
J
Ludwig
Alexander
A
Nieß
Martin
M
Siemann-Herzberg
D004926:Escherichia coli / Q000502:physiology*; D008856:Microscopy, Fluorescence; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000502:physiology*
D012335:RNA, Ribosomal; D012269:Ribosomal Proteins
capillary gel electrophoresis; fluorescence; growth rate; rRNA; ribosomal protein; ribosome degradation; ribosome dynamics