Working at temperatures near absolute zero, UC Berkeley experts in electron microscopy have learned in detail how proteins orchestrate the first key steps in gene activation – opening up the double-stranded DNA.
There would be no living cells, let alone humans, if the two DNA strands making up double-helix molecules of DNA remained forever entwined. To switch on a gene requires the separation of the strands, followed by transcription of one strand to make messenger RNA.
The RNA travels to the cell’s protein factory, where it serves as a template for cranking out specialized proteins based on the genetic code. Collectively, these proteins, made at the proper time and place by cells throughout the body, guide development, do the cells’ work, send signals and provide structural support. When this finely orchestrated assembly line is disrupted or diverted, the result is cellular malfunction and disease.
Eva Nogales, a professor of biochemistry, biophysics and structural biology at UC Berkeley who led the new study, aims to further explore the interactions between these key proteins and DNA, and perhaps to gain insight into how these might go awry in disease, leading specific genes to be switched on or off abnormally. “That’s the value of basic research,” she said. “Without knowing how the machine normally works, you won’t really know how it breaks down and how to fix it.”
“The iconic DNA structure that so many know and love and that we now see in many logos has to open up,” said Nogales, who is a Howard Hughes Medical Institute (HHMI) investigator as well as a senior faculty scientist at the Lawrence Berkeley National Laboratory.
But while the double helix is a familiar structure, until now much less has been understood about how the double helix opens up to form a “transcription bubble” that enables messenger RNA to be made from a DNA template at specific points along the double helix where gene expression is initiated.
In a tour-de-force of structural biology published online in Nature on May 11, Nogales’ team used cryo-electron microscopy — freezing purified samples and photographing them with electrons instead of light — to obtain data and images of the human molecular machinery engaged with DNA at a near-atomic level of resolution. This is the greatest level of detail ever obtained for the “pre-initiation complex” of human proteins that launches RNA production.
This biological interaction could not be more fundamental to life, and by obtaining such detailed structural information about a vital event that each cell must carry out thousands of times each second, Nogales also has laid the groundwork for studying interactions between specialized regulatory proteins that influence the activation of specific genes by interacting with DNA and the pre-initiation complex.
According to study co-author Robert Tjian, a leading expert in gene regulation, president of HHMI and professor of biochemistry, biophysics and structural biology at UC Berkeley, “This work achieves a longstanding grand challenge that has been pursued for decades, and 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.”
Yuan He, a former postdoctoral fellow working in Nogales’ lab and now an assistant professor at Northwestern University, carried out the electron microscopy experiments, which included imaging the human pre-initiation complex in three different purified states representing sequential points in time: the closed state engaged with DNA in the double helix; an open state engaged with the transcription bubble, and a DNA/protein complex in which transcription of RNA had begun.
The new electron microscopy data the researchers obtained included the first structural visualization of the full transcription bubble as it is opened by the pre-initiation complex, and the detailed tracking of movements for some of the key proteins in this process. The study authors combined new information with previously obtained structures of some individual protein components to model the entire structure of the complex and its transitions through the three states. The results can be viewed in a computer animation also published online in Nature.
The events detailed by the research team occur similarly in diverse organisms as simple as yeast and as complex as humans, and even in bacteria. One might assume that genetic mutations in such a vital structure would be disastrous, and in humans, mutations in the best-known, textbook enzyme at the heart of the pre-initiation complex — the massive enzyme called RNA polymerase II — are very likely to be lethal before birth.
But for many of the proteins involved in the pre-initiation complex, nature through evolution has messed with the formula a bit, with the human system and associated protein structures often being more elaborate than they appear in our simplest evolutionary ancestors, such as bacteria, in which the complex is much smaller and has many fewer components. In general these differences have resulted in higher organisms such as humans having very tight regulation to determine exactly where, when and how often the pre-initiation complex will bind to DNA to ensure that the correct sequence and amount of RNA is transcribed.
One aspect of messenger RNA transcription seems haphazard for such a vital process. The making of a complete RNA transcript typically is preceded by several false starts that produce only very short strands of RNA, Nogales said. This is because the enzymes in the complex that assist RNA polymerase II by holding the DNA open in the transcription bubble must later drop off when they are no longer needed to allow transcription to go forward to completion. Oftentimes their timing is off.
“In evolution, the result does not have to be perfect,” she said. “It just needs to get the job done well enough to allow the organism to survive.”