Single Molecule Study of Rela During the Stringent Response in Live E. Coli Cells

Abstract

The stringent response is a physiological response that occurs when bacterial cells encounter nutritional stresses such as amino acid starvation or fatty acid starvation. The most marked outcome of this response is an immediate accumulation of the effector nucleotides, guanosine tetra- and pentaphosphate (ppGpp and pppGpp).The RelA protein of Escherichia coli is a (p)ppGpp synthetase that is activated by amino acid starvation. Here, we use single molecule tracking method (sptPALM) to investigate the RelA protein association and dissociation behavior before and after the stringent response. In contrast to the earlier work in which RelA was found to diffuse like ribosomes in normal growth conditions and to diffuse freely following the stringent response, we find RelA diffusion under both conditions to be heterogeneous. And during the stringent response, RelA diffuses more slowly than in the normal growth condition. In addition, the fraction of the slow diffusion population increases. This indicates that during the stringent response, RelA tends to bind to ribosomes more often compared to the normal growth condition, which suggests that ribosomes need to be on ribosomes to synthesize (p)ppGpp.