Structural Changes in the E. coli Ribosome During Assembly and Cellular Stress

Ribosome biogenesis;Cellular stress;Escherichia coli;High-throughput sequencing;RNA probing;Biophysics

Hulscher, Ryan MartinWoodson, Sarah A ;

Johns Hopkins University

关键词: Ribosome biogenesis;    Cellular stress;    Escherichia coli;    High-throughput sequencing;    RNA probing;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60234/HULSCHER-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The ribosome is a large ribonucleoprotein that undergoes structural changes throughout its lifecycle. In this work, I will present a method that I developed for pulse-labeling E. coli ribosomal RNA. This procedure was coupled with RNA probing techniques for the structural analysis of in vivo assembly intermediates. This method was able to identify global structural changes in the ribosome structure during assembly.I also used RNA probing techniques to study structural changes that occur as the E. coli ribosome is degraded under bacterial stress. Cells that were grown in rich media to late stationary phase contained rRNA that was more accessible to DMS structure probing, suggesting increased disorder and the potential for degradation by cellular nucleases. The results of the DMS probing showed how cellular nuclease cleavage sites were more accessible in stressed cells. This change in the accessibility of the rRNA correlates well with the decrease in intracellular rRNA that I observed over the same time period.The polysome profiles of stressed bacterial cells show that deprivation of different nutrients can result in different responses in how ribosome degradation is regulated. Ribosomes in cells deprived of magnesium will be degraded in a matter of hours, whereas nitrogen-starved cells still have ribosomal particles after 4 days of nitrogen depletion. The MRE600 cells used for the experiments maintain ribosomes during bacterial stress without forming the 100S hibernation ribosome.