Pushing the limits of cryo-electron microscopy, scientists have captured freeze-frames of the changing shape of a huge molecule, one of the body's key molecular machines, as it locks onto DNA and loads the machinery for reading the genetic code.

The molecule, called transcription factor IID, is critical to transcribing genes into RNA that will later be used as blueprints to make proteins. Because of its many moving parts and large size, however, TFIID's 3D structure has been hard to capture: the moving parts become a blur.

Cryo-EM, an imaging technique whose discoverers garnered the 2017 Nobel Prize in Chemistry, is the only way to obtain a snapshot of bulky, floppy structures like this. High-resolution structural information is essential for understanding how TFIID translates the operating instructions in our genome and how it sometimes goes haywire.

The new, more detailed snapshots of the molecule's moving parts could help drug designers create drugs that interfere with the molecule's structural changes in order to tweak the expression of a gene that is causing disease. Researchers posted their findings in the journal Science.

TFIID is an agglomeration of more than a dozen distinct proteins that homes in on a promoter - a region of DNA that controls the transcription of a nearby gene - and tests the sequence to make sure it has landed at the right spot. Once this is confirmed, it opens up to recruit dozens of other proteins that then start ratcheting along the gene, using the DNA sequence as a template to create a complementary sequence of RNA, called messenger RNA. This then wends its way out of the nucleus into the body of the cell, where it is translated by other molecular machines into protein.

Cryo-EM involves freezing a drop containing millions of copies of a molecule, in every imaginable orientation, and using an electron microscope to determine the structure by combining images to define the 3D shape. Because TFIID has many moving parts as it binds to DNA and gets ready to transcribe a gene, averaging all the frozen positions produces a blurred image.

The improved pictures are a result of better detectors developed and steadily improving computer algorithms to analyze the huge amounts of data collected by the detectors. This helped the team to define five distinct structures of the TFIID molecule.

They span the whole binding sequence: before binding to the DNA, initial binding to the promoter, subsequent binding after it double checks that this is the right place, and the final state," the senior author said.

https://news.berkeley.edu/2018/11/16/freeze-frame-microscopy-captures-molecules-lock-and-load-on-dna/

http://science.sciencemag.org/content/early/2018/11/14/science.aau8872?rss=1