Your DNA governs more than just what color your eyes are and whether you can curl your tongue. Your genes contain instructions for making all your proteins, which your cells constantly need to keep you alive. But some key aspects of how that process works at the molecular level have been a bit of a mystery—until now.
Using cryo-electron microscopy (cryo-EM), Lawrence Berkeley National Laboratory (Berkeley Lab) scientist Eva Nogales and her team have made a significant breakthrough in our understanding of how our molecular machinery finds the right DNA to copy, showing with unprecedented detail the role of a powerhouse transcription factor known as TFIID.
Berkeley Lab scientists Eva Nogales and Robert Louder at the electron microscope. (Credit: Roy Kaltschmidt/Berkeley Lab)
This finding is important as it paves the way for scientists to understand and treat a host of malignancies. “Understanding this regulatory process in the cell is the only way to manipulate it or fix it when it goes bad,” said Nogales. “Gene expression is at the heart of many essential biological processes, from embryonic development to cancer. One day we’ll be able to manipulate these fundamental mechanisms, either to correct for expression of genes that should or should not be present or to take care of malignant states where the process has gone out of control.”
“We now have the structure of the whole protein organization that is formed at the beginning of every gene. This is something no one has come close to doing because it is really very difficult to study by traditional methodologies.”
How genetic information flows in living organisms is referred to as the “central dogma of molecular biology.” Cells are constantly turning genes on and off in response to what’s happening in their environment, and to do that, the cell uses its DNA, the big library of genetic blueprints, finds the correct section, and makes a copy in the form of messenger RNA; the mRNA is then used to produce the needed protein.
The problem with this “library” is that it has no page numbers or table of contents. However, markers are present in the form of specific DNA sequences (called core promoter motifs) to indicate where a gene starts and ends. So how does the polymerase, the enzyme that carries out the transcription, know where to start? “DNA is a huge, huge molecule. Out of this soup, you have to find where this gene starts, so the polymerase knows where to start copying,” Nogales said. “This transcription factor, TFIID, is the protein complex that does exactly that, by recognizing and binding to DNA core promoter regions.”
What Nogales and her team have been able to do is to visualize, with unprecedented detail, TFIID bound to DNA as it recognizes the start, or promoter, region of a gene. They have also found how it serves as a sort of landing pad for all the molecular machinery that needs to assemble at this position—this is called the transcription pre-initiation complex (PIC). This PIC ultimately positions the polymerase so it can start transcribing.
The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1–13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP–TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.
SOURCES – Nature, Berkeley National Lab