Protein Takes on New Job, and Scientific Dogma, by Refolding
By Anette Breindl
The notion that one gene can make only one protein, once a central tenet of molecular biology, has long since been revised. One gene can make several proteins through alternative splicing.
Now, scientists have discovered another way that several proteins can result from the same gene. In the July 20, 2012, issue of Cell, they reported that the transcription factor RfaH can morph into a translation initiation factor by refolding a large part of itself into a completely new shape. The work may enable the design of protein switches that can change their functions in response to changing cellular conditions.
The researchers did their experiments in E. coli. But "we are talking here about a highly conserved protein," Irina Artsimovitch told BioWorld Today. "In fact, it is the only conserved transcription factor." Artsimovitch is a professor of microbiology at Ohio State University and, with Paul Roesch, co-corresponding author of the paper describing the findings.
RfaH consists of two subdomains that separately fold into distinct shapes, and are joined by a flexible linker. Researchers had worked out that it functions as a transcription factor. The two parts of the protein interact with each other, and with RNA polymerase, which transcribes messenger RNA off of DNA. When RfaH is bound to the polymerase, it increases transcription because the RNA polymerase, which works in something of a stop-and-go fashion, pauses less.
Artsimovitch and her team made their unexpected discovery in the time-honored way of science: through being thoroughly confused about their data.
Artsimovitch explained that "initially, we assumed" – reasonably enough – "that [RfaH] had the same structure as its closest homolog," the transcription factor NusG. But when they tried to determine the structure experimentally starting from that assumption, "we could not get any meaningful structure from the data."
Instead, they ultimately realized that the structure of RfaH was "dramatically different – as different as it could be" from its homolog. Or at least, the two structures are dramatically different when the two subdomains of the protein are interacting.
But when RfaH binds to its polymerase target, the two parts of the protein no longer interact. One half of the protein is essentially left to its own devices, though still joined to the rest of the protein by the linker region.
Under those circumstances, the freed-up part of the protein refolds into a completely new shape – in this case, a shape that does closely resemble its NusG homolog.
And in experiments, the team demonstrated that with the new shape came a new binding partner. Once it was refolded, the free domain bound to the ribosome, the machinery of the cell that translates protein off of messenger RNA. Such binding massively increased translation, by up to a thousandfold.
The alternative folding possibilities of RfaH are somewhat similar to those of prions, which also can fold into two different shapes. But in the case of prions, one of those shapes is bad news for the cell – it leads to aggregates that are the cause of neurodegenerative disease. In contrast, RfaH has critical functions in each of its shapes. In fact, another practical aspect of the work besides protein switches might be in harnessing how strongly it affects translation for increasing the levels of certain proteins in cells.
The reason folded proteins stay stable once they have folded is that a folded state has lower energy than an unfolded one. To unfold and refold, RfaH needs to pass through a high-energy state. How that happens is still unclear to Artsimovitch and her team.
One possibility is that the two folding patterns are in equilibrium, and as the protein binds to RNA polymerase, the concentration of the first pattern is increased, pushing that equilibrium toward generation of the second folding pattern. Another is that interactions with the ribosome alter the protein to make its refolding possible. And molecular chaperones help fold, and in some cases unfold, many different proteins.
Another surprising aspect of RfaH's switcheroo is that it ever makes it into its first conformation in the first place. While that shape is lower energy than an unfolded protein, it is higher energy than the shape it morphs into. "From the energetics," Artsimovitch said, "you would predict the refolded state."
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