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Researchers Watch RNA Folding


BioTechniques

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October 18, 2012

In a soundproof, temperature-stabilized, vibration-controlled basement laboratory, researchers at Stanford University observed RNA transcription in real-time, ogling as a single nascent strand of RNA grew—nucleotide by nucleotide—and folded into a regulator riboswitch.

“Until now, no one has actually seen, at the single molecule level, an RNA fold with functional consequences,” said Steven Block, a biophysicist at Stanford University and author on the study, published October 19 in Science. “This is the first time anybody has watched it as it’s being made.”

To accomplish this feat, Block and his colleagues developed an optical trapping assay, so-called optical tweezers, using two plastic beads and two beams of laser light to measure the transcription process. The technique is an improvement over conventional, indirect methods for studying RNA folding, such as computer modeling, denaturing fully formed RNA, or studying the actions of riboswitches in bulk.

In their optical tweezer approach, one bead was attached to one molecule of RNA polymerase connected to the DNA template for the pbuE riboswitch, which regulates transcription of a Bacillus subtilis gene that moves adenine out of a cell. The other bead was attached to a DNA handle with a short, single-stranded sequence at the end complementary to the transcribed RNA, which held the transcribed RNA.

Like two tractor beams, the streams of photons exerted a slight force onto the plastic beads, holding them in place and allowing the researchers to tug on the RNA molecule and measure the strand as it elongated and folded at sub-nanometer lengths. Using scattered laser light and computer sensors, they precisely visualized the RNA’s extension on Block’s computers.

“As the RNA is being made one nucleotide at a time, intermediate, perhaps unstable structures can form that are not present in the final form,” said Block. Those structures can have important functional consequences.

In this case, as the riboswitch is transcribed, it starts to fold into an aptamer. The aptamer is unstable, and it quickly collapses into a terminator sequence that halts the transcription of the gene. But if a lot of adenine is present, adenine binds the aptamer. This stabilizes it temporarily, prevents the formation of the terminator, and allows transcription of the rest of the gene to continue, creating a protein that will eventually shuttle adenine out of the cells.

In this work, Block and colleagues discovered that this aptamer configuration lasts just seconds—the aptamer is 10 billion times less stable than the terminator sequence. This is just long enough to transcribe the gene pbuE, and the aptamer soon re-configures into a terminator. Without real-time single molecule visualization, scientists would never see the aptamer or understand how it regulates a gene.

Now, Block plans to continue using the technique to study different adenine riboswitches in real-time, as well as the movement and folding of several other small molecules. “Understanding how RNA folds is absolutely fundamental to understanding molecular biology. RNA is probably the first coded molecule to come along: it can fold and make enzymes, it has catalytic activities, it can control genes,” said Block.

Reference

1. Frieda, K. L. and S. M. Block. 2012. Direct observation of co-transcriptional folding in an adenine riboswitch. Science (October 19).


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