Life-creating complex in stunning resolution
Image: Professor Eva Nogales and colleagues unravel structure of human pre-initiation complex.
Using cryo-electron microscopy, researchers from the Lawrence Berkeley National Laboratory have captured near-atomic resolution images of the human proteins that launch RNA production, a biological event fundamental to human life.
Obtaining the structural detail of this protein machinery will help researchers study the many genetic defects that can lead to human disease.
"Without knowing how this machine normally works you won't really know how it breaks down and how to fix it," says Professor Eva Nogales, a Howard Hughes Medical Institute investigator. "That's the value of basic research,"
DNA unfolding to make RNA. RNA appears briefly in bright red. (Credit: Eva Nogales)
The initiation of DNA transcription involves complex interactions between RNA-synthesizing RNA polymerase (Pol) enzymes, transcription factors and DNA.
A large pre-initiation complex (PIC) is required to open up the double helix DNA and ultimately trigger gene expression but the sheer size and flexibility of the unit has presented massive challenges for structural biologists attempting to study the complex.
With this in mind, Nogales and colleagues loaded purified PIC samples onto EM grids, plunge-freezing these in liquid ethane.
The frozen samples were then imaged using a FEI Titan EM operating at 300 kV, with a Gatan K2 summit direct electron detector.
For each cryo-EM dataset generated, the researchers collected between 855 and 1334 movie series.
They then used image processing software to select hundreds of thousands of particles to classify and reconstruct 3D structures as well as eventual PIC atomic models.
According to the researchers, they obtained data and images of the complex in three sequential states at near-atomic resolution, as high as 3.9 Å.
This is greatest level of detail ever obtained for the pre-initiation complex of human proteins, and as Nogales' colleague Professor Robert Tjian highlights: "This work has been pursued for decades."
"These remarkably high-resolution structures of the protein machinery engaged in transcribing DNA will have profound implications for deciphering how mistakes in gene control leads to many human diseases," he adds.
Research is published in Nature.