Water Molecules Play a Vital Role in Gene Transcription

While scientists have studied the molecular steps involved in gene expression for decades, many questions still remain about this fundamental process. To transcribe genes, RNA polymerase II (RNA Pol II) binds to DNA and catalyzes the addition of nucleotides to a growing mRNA chain. The precise molecular details of how RNA Pol II adds nucleotides to mRNA, though, remain unclear.

To find out, a collaborative team of researchers led by Dong Wang, a researcher studying DNA damage responses and DNA modifications’ effect on transcription at the University of California, San Diego, used cryogenic electron microscopy (cryo-EM) to study the arrangements of RNA Pol II and its substrates before and after its catalytic activity in high-resolution. They published their findings in Molecular Cell, showing that individual water molecules interact with RNA Pol II and other substrates during transcription in Saccharomyces cerevisiae.¹ The results revealed the mechanistic processes behind the first steps of gene expression and support a reimagining of the role of water in biochemical processes.

Prior to the present study, previous work had shown that water contributed to the stability and structure of several molecular processes.^2-4 However, studying water’s role in RNA Pol II catalytic reactions has been challenging to do with traditional structural methods like cryo-EM because of the need for very specific orientations of the components and reducing air-water interfaces. To overcome this, Wang and his team used an approach developed by coauthor Peijun Zhang, a biophysicist at the University of Oxford. This strategy leveraged streptavidin/biotin binding to tether the researchers’ target molecules in a single layer on the cryo-EM surface, allowing them to reduce unwanted exposures and complex orientations.⁵ Using this approach, the team was able to obtain two high-resolution images of RNA Pol II associated with its substrates and water molecules before catalysis and one image of these molecules after.

In RNA Pol II’s pre-catalytic state, the researchers saw 1,357 water molecules that promoted either protein-protein or protein-nucleic acid interactions. Some water molecules helped align two magnesium ions and adenosine triphosphate, while another water molecule helped distinguish between ribose and deoxyribose. Chains of water molecules also helped bridge phosphates to specific residues in the active site of RNA Pol II.

Besides serving as scaffolding between substrates, the water molecules also resided at sites where they could act as proton donors during the addition of nucleotides to the growing mRNA chain. The researchers showed that another water molecule acts as a proton acceptor to deprotonate the 3’ hydroxyl group of the RNA primer during this process.

Additionally, the researchers hypothesized that hydrogen bonds between water molecules and nucleic acids in the transcription bubble can stabilize the DNA-RNA hybrid and act as a “lubricant” to help RNA Pol II move along the DNA during transcription.

In the post-catalytic state, Wang and his coauthors identified 712 water molecules involved in stabilizing the structure. Although many of the molecules were in the same positions as in the pre-catalytic orientation, some water molecules shifted to replace the space of the nucleotide triphosphate after the chemical reaction.

“Our work unveils unprecedented and critical roles of water molecules in substrate recognition and the catalytic mechanism of transcription,” the authors wrote in their study. They also referenced an unrelated study assessing the role of water in gene transcription in Escherichia coli, which detailed similar findings.⁶ “The elucidation of a complete active site of transcription machinery reveals that functional waters are evolutionarily conserved and integral components of transcription machinery with critical roles in transcription catalysis, which marks a major conceptual leap beyond the traditional ‘‘protein-centered’’ paradigm of transcription,” the authors wrote about these coinciding results.