Similar to how congested environments impact how humans move through space, crowding at the molecular level impacts how biomolecules behave. A new study published in the Proceedings of the National Academy of Sciences indicates that crowded conditions can have a dramatic impact on the rates at which biomolecules fold into their mature forms. This research has important implications for both understanding biomolecular behavior, and also for helping scientists to better replicate cellular conditions for future experimentation.
Just like the human body, the cells of an organism (human or otherwise) are composed of many diverse and complex molecules in a largely watery environment. The amount of water within a cell can vary considerably based on the cell type and the environment in which that cell lives. Within these watery structures, it is said that “Cells are 25 to 35% filled with ‘stuff'”, with “stuff” referring to proteins, nucleic acids, lipids, and other important molecules that the cell needs to survive.
Researchers hypothesized that having all this “stuff” in a cell might contribute to different behavior in how biomolecules form and are further modified. One of the molecules of particular interest with respects to the impacts of biomolecular crowding is RNA. RNA is a 3D molecule that is essential for creating new proteins and/or acting as an enzyme to catalyze chemical reactions. Like many of the molecules within a cell, RNA is first made in a long chain-like molecule that must then fold into its correct 3D structure. Therefore any effects created by biomolecular crowding on RNA folding could potentially have a great influence on a cell.
To study the effects of biomolecular crowding on RNA folding, a team of researchers from the Joint Institute for Lab Astrophysics (JILA) and the National Institute of Standard Technology (NIST) sought to compare the differences between the rates of RNA folding between dilute “test tube” conditions and more concentrated “cellular conditions.” They did this using a new research technique, called “single molecule fluorescence technology,” which makes unfolded RNA molecules glow green and folded RNA molecules glow red. They simulated the crowded intra-cellular conditions by using the long, chain-like molecule polyethylene glycol (PEG)– an ingredient in antifreeze. PEG naturally expands to take up a lot of room, but does not in any other way interact with RNA molecules.
The researchers found that on average, single RNA molecules will fold 35 times faster in the crowded cellular conditions than in the conditions that mimicked the dilute test-tube environments. In addition, the rate of RNA unfolding decreased.
These findings have important implications for both understanding the inner workings of cellular biology, but also in offering an explanation as to why RNA (and perhaps other molecules as well) are so difficult to study outside of the cell. It is possible that one reason why in vitro studies (studies outside of the living unit—in this case the cell) have faced difficulties in reproducing their results in in vivo experiments (experiments within the living unit—in this case the cell) is that the in vitro studies do not adequately crowd the molecules as they would naturally be within a cellular environment.
The next step for the JILA/NIST researchers will be to study RNA folding rates in a soup of broken-up bacteria. The bacterial soup will mimic internal cellular environments even more closely, and shed further light on biomolecular crowding. And beyond that, it is hoped that one day a method will be devised to study RNA folding as it naturally happens within a living, crowded cell.
By Sarah Takushi