RNA polymerase II transcription complexes were isolated directly from fruit fly embryos, preserving many of the proteins, DNA, RNA and nucleosomes present in the cell. Cryo-electron microscopy produced thousands of images and computational analysis sorted the imaging data into distinct groups to reconstruct multiple 3D-dimensional structures. The novel approach revealed that transcription complexes inside cells are not all identical, but instead exist in several structural forms.
Image Credit: Courtesy of Katsuhiko Murakami / Pennsylvania State University
(CC BY-NC-ND 4.0)
Scientific Frontline: Extended "At a Glance" Summary: Native Gene Transcription Complexes
The Core Concept: Researchers have successfully isolated and observed the nanoscale machinery responsible for gene transcription (eukaryotic RNA polymerase II) operating in its natural, unpurified state inside living cells.
Key Distinction/Mechanism: Prior to this study, RNA polymerase II was primarily observed in highly controlled, artificial laboratory conditions (in vitro), where it was assumed to consist uniformly of 12 subunits; however, observing it in its native state (in vivo) revealed a dynamic mix of structures, with some complexes unexpectedly missing two subunits.
Origin/History: The foundational idea for this specific methodological approach originated in 2021 when David Gilmour presented partially purified RNA polymerase II extracted from a fruit fly embryo to Katsuhiko Murakami, leading to the current findings published in Nature Communications.
Major Frameworks/Components:
- Eukaryotic RNA Polymerase II: The specific enzyme complex responsible for copying DNA instructions into RNA.
- Cryo-Electron Microscopy (cryo-EM): An advanced imaging technique utilized to freeze and visualize the transcription complexes at near-atomic resolution.
- Transcription Complexes: The intact clusters of RNA polymerase II, DNA, RNA, and associated proteins involved in the gene-reading process.
- Fruit Fly Embryos (Drosophila melanogaster): The specific biological organism used to extract the native transcription complexes.
Branch of Science: Molecular Biology, Biochemistry, and Structural Biology.
Future Application: This novel methodology paves the way for observing other complex cellular processes in their natural environments across various organisms, including archaea, potentially providing more accurate blueprints of cellular function for medical and pharmacological applications.
Why It Matters: By capturing the "messy" reality of cellular processes as they occur naturally, rather than in sanitized, isolated lab environments, this research marks a significant shift in modern biology toward understanding the true structural diversity and dynamics of life at the molecular level.
All living things have a fleet of tiny copy machines that turn instructions from DNA into RNA, the molecules responsible for helping build proteins that keep organisms alive. One of these nanoscale machines, called eukaryotic RNA polymerase II, performs a crucial early step required by nearly every biological process. It creates messages, copying and carrying instructions from the cell nucleus to synthesize proteins.
Until recently, scientists had only witnessed eukaryotic RNA polymerase II at work in carefully assembled test tubes, stripped of the chaotic realities of life inside a cell. Now, a team led by Penn State researchers has captured a glimpse of this molecular machinery as it operates inside living organisms. They reported their findings in the journal Nature Communications.
“This is the first time we’re seeing this process as it actually happens, the way it acts when no one is watching,” said Katsuhiko Murakami, the Stanley Person Professor of Molecular Biology and director of the Huck Center for Structural Biology at Penn State, and co-corresponding author of the study. “Previously, we had to use highly purified samples under ideal lab conditions to visualize their structures, which is not how life really works. Now, we must completely change our thinking, because what we’re seeing is not anything we have seen before.”
Using fruit fly embryos, the team developed a method to extract intact “transcription complexes,” the clusters of RNA polymerase II and DNA involved in reading and copying genes. They then used cryo-electron microscopy (cryo-EM), a powerful imaging technique that freezes molecules in place and visualizes them in near-atomic detail, to map their findings. What emerged was a far more dynamic and surprising picture of the copying process, called gene transcription, than they had expected.
Murakami explained that the project began in 2021 when David Gilmour, emeritus professor of biochemistry and molecular biology at Penn State, showed him purified RNA polymerase II extracted from a fruit fly embryo.
“It wasn’t clean, but it sparked an idea,” he said. “We could use cryo-EM to analyze native transcription complexes from it. After years of hard work, we’ve now captured these complexes in a near-native state, and what we found was actually pretty surprising.”
Prior to this study, RNA polymerase II was thought to comprise twelve subunits that come together to form one complete unit, so most researchers assumed all complexes looked the same in cells, he said. However, the team’s results showed this is not always the case. Some complexes possess all twelve subunits, as expected, but others lack two subunits, leaving them with only ten.
“That kind of variation wasn’t what people thought would happen, so it was a really unexpected finding,” Murakami said.
The discovery may offer clues to how cells manage the delicate balance between tightly packing DNA and making it accessible when needed. Such moments of transition are difficult to observe in traditional experiments, he said, making their appearance in the team’s cryo-EM imaging study particularly significant.
“From a basic science perspective, the approach used to be pretty straightforward,” said Jean-Paul Armache, assistant professor of biochemistry and molecular biology at Penn State and co-author of the paper. “Researchers would try to capture one clear picture of something, study what they saw, and then publish their findings. Now, instead of looking at just one controlled version, we can study a mix at the same time and understand how they vary. That gives us a much fuller, messier, and more accurate picture of what’s going on. We’re not seeing one sanitized process; it’s everything simultaneously—how life really happens.”
The study is part of a broader shift in modern biology, Armache said, with researchers moving away from laboratory-built systems and toward observing molecules as they exist in living cells. A better understanding of how the gene-reading process works in real-life conditions offers a more accurate blueprint of cellular function, which can have applications in fields such as medicine, he said.
The team’s approach could open the door to studying many other complex cellular processes in their natural environments, Murakami said.
“We’re starting to use this system more broadly, not just in one kind of organism, but also in others, such as archaea, which are part of the microbiota of all organisms,” Murakami said. “We’re trying to understand how these molecules behave in real conditions and different environments, so this is just the beginning of what we’ll be able to see.”
Funding: This work was supported by the Pennsylvania Department of Health and the National Institutes of Health under award numbers R35 GM156623, R21AI168948, R01 GM047477, R01 GM098399, and R35 GM161751.
Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Published in journal: Nature Communications
Authors: Natalie L. Venette-Smith, Rishi K. Vishwakarma, Varun Venkatakrishnan, Roberta Dollinger, Josie Schultz, Paul Babitzke, Ganesh Anand, David S. Gilmour, Jean-Paul Armache, and Katsuhiko S. Murakami
Source/Credit: Pennsylvania State University | Adrienne Berard
Edited by: Scientific Frontline
Reference Number: mbio082126_01