The researchers uncovered clues that may explain how and why a particular virus, called N4, injects an unusual substance -- an RNA polymerase protein -- into an E. coli bacterial cell. In this cryo-electron micrograph image, an N4 phage virus is shown sitting on the surface of a bacterial cell. It is injecting a molecule of DNA (shown in red, yellow, and blue) and four RNA polymerase proteins (shown in blue) into the bacterial cell. In addition, the bacterial cell's DNA (shown in gray) is visible at the bottom of the image. Credit: K.H. Choi and J.D. Hayden, Penn State
It turns out that the N4 RNA polymerase is able to respond only to DNA that is shaped like a hairpin. Part of the N4 phage's DNA is shaped like a hairpin, whereas the E. coli bacterium's DNA is not shaped like a hairpin. Once the N4 RNA polymerase interacts with the phage's hairpin DNA, it begins to change its shape from a fisted form to a cupped form. By opening up, the RNA polymerase exposes its active site, which allows it to begin the transcription process.
While the researchers determined that the N4 RNA polymerase must change its form in order to bind to the phage DNA, they also found that this transformation isn't the polymerase's first as it progresses through the steps of phage infection. The team found that the polymerase must change form in order to squeeze through the phage's tiny injection tube as it is injected into the E. coli cell. "The diameter of the tube is narrower than the diameter of RNA polymerase," said Murakami. "This means that the enzyme must be unfolded into a longer and thinner structure in order to fit through the tube, and then it is refolded after it is injected into the cell."
The ability of the N4 RNA polymerase to withstand this unfolding and refolding is unique. Therefore, the team decided to experiment with this property by exposing the polymerase to high temperatures. As expected, the high temperatures caused the molecule to unfold. The scientists then cooled the molecule and watched as it reformed into its original shape
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