How individual RelA enzymes sense starvation in a living cell

We have combined biophysical and imaging techniques to study individual enzyme molecules in living bacterial cells with millisecond temporal resolution and super-resolution spatial precision. We have applied this method to elucidate the mechanism of the regulatory enzyme RelA, which plays a key role in the starvation adaptation mechanism, the stringent response.

HFSP Cross Disciplinary Fellow Brian English and colleagues
authored on Wed, 20 July 2011

How enzymes function inside the living cell remains an important question in enzymology. Here we study the enzyme RelA, which plays a central role in the starvation adaptation mechanism, called the stringent response. During this response, RelA synthesizes a small messenger molecule ppGpp, which globally rewires the bacterial cell on transcriptional, translational and replicational levels. The catalytic mechanism of RelA is still a matter of debate in the literature, but is known to involve interaction with the protein synthesizing machinery, the ribosome. Binding to the ribosome is expected to slow down the diffusion of RelA dramatically, and we use this change as a readout of its catalytic cycle.

Figure: When E. coli cells are growing exponentially, inactive RelA is tightly associated to the ribosome (left panel). A typical diffusion trajectory of RelA with a frame time of 20 ms is displayed below. When cells are severely starved of amino acids (right panel), uncharged tRNA binds to the A site of the ribosome. Active RelA unbinds from the ribosome, and fast RelA diffusion trajectories are observed (below, with 20 ms frame time).

Here we combine single molecule tracking under stroboscopic illumination and photoconversion. This combination allows for reliable detection of fast and freely diffusing individual target molecules in the bacterial cytoplasm irrespective of their cellular copy numbers. Based on our in vivo approach, we propose an ‘extended hopping model’ for the mechanism of RelA: We show that dormant RelA is tightly associated with the ribosome in non-starved cells, and that it dissociates from the ribosome upon starvation. Furthermore, upon severe amino acid starvation conditions, active RelA remains dissociated for an extended period. A much more transient response is observed during heat-shock, where RelA quickly rebinds to the ribosome.

This method can be applied to any intracellular reaction that involves target search, be it protein interactions with DNA, RNA, membranes, or protein complexes, as binding to these targets will necessarily involve changes in the diffusion characteristics of the enzyme of choice. Suitable in vitro systems are often lacking in hub enzymes that may interact with a multitude of binding partners, and where potential binding partners may yet be unknown. A direct mechanistic study of such regulatory enzymes is now feasible.

Reference

Single Molecule Investigations of the Stringent Response Machinery in Individual Living Bacterial Cells. BP English, V Hauryliuk, A Sanamrad, S Tankov, NH Dekker, and J Elf. Proceedings of the National Academy of Sciences (2011), doi: 10.1073/pnas.1102255108.

Pubmed link

PNAS link

... and translated into Basque!