An international team of researchers has utilized cutting-edge microscopy techniques to observe how ribosomes attach to mRNA.
In cellular biology, DNA serves as the blueprint for protein construction.
To synthesize proteins, cells first transcribe DNA into a copy known as mRNA. This is then read by ribosomes, which transform the mRNA sequence into protein. However, the process of how ribosomes connect to and interpret mRNA has remained elusive until now.
Recently, a collaborative group of scientists, including researchers from the University of Michigan, leveraged advanced microscopy to visualize the recruitment of ribosomes to mRNA while it is being transcribed by an enzyme known as RNA polymerase (RNAP). Their findings, focusing on the transcription process in bacteria, are detailed in the journal Science.
“Grasping how the ribosome engages with or ‘recruits’ the mRNA is essential for understanding the subsequent steps, like how it decodes the information within the mRNA,” explained Albert Weixlbaumer from the Institut de génétique et de biologie moléculaire et cellulaire in France, who co-led the study. “It’s akin to having a book that you need to read and interpret, but you have no idea how to obtain it. How is the book delivered to the reader?”
The research team found that during transcription, RNAP employs two distinct anchors to effectively secure the ribosome, ensuring a strong initiation of protein production. This process is comparable to a supervisor at a construction site making sure that all components are assembled correctly to provide optimal stability and functionality.
According to the researchers, mastering these basic biological processes may lead to new antibiotic developments that specifically target bacterial protein synthesis pathways. While antibiotics have typically focused on the ribosome or RNAP, bacteria often adapt and develop resistance. With their new insights, the team aims to outsmart bacteria by disrupting multiple pathways.
“We understand there is an interplay involving RNAP, the ribosome, transcription factors, proteins, and mRNA,” said U-M senior scientist Adrien Chauvier, one of the four co-leaders of the study. “We could focus on this interaction point, specifically between RNAP, the ribosome, and mRNA, with a molecule that disrupts the recruitment or stability of the assembly.”
The team established a framework to illustrate how the various elements of the complex work together to transport newly transcribed mRNAs to the ribosome, thus bridging the transcription and translation processes.
“We wanted to understand how the connection between RNAP and the ribosome is initially formed,” Weixlbaumer commented. “By using purified components, we recreated the complex—measuring a mere 10 billionths of a meter in diameter—and observed their actions with cryo-electron microscopy (cryo-EM). We then needed to determine if the behaviors we noted in our purified components could be replicated in other experimental systems.”
In more intricate human cells, DNA is sequestered within a nucleus, where RNAP functions as the “interpreter,” breaking genetic information into manageable pieces. This powerful enzyme transcribes the DNA into mRNA, which serves as a selected copy of a segment of the genetic code that is then moved into the ribosome located in the much more spacious cytoplasm, where it is turned into proteins, the fundamental building blocks of life.
In prokaryotes, which lack a defined nucleus and internal membrane structures, transcription and translation occur simultaneously and very close to one another. This allows RNAP and the ribosome to directly coordinate their functions.
Bacteria, being the most well-understood prokaryotes due to their simple genetic framework, provided an ideal model for the team to investigate how the ribosome and RNAP connect during gene expression.
The researchers applied a variety of technologies and methods according to each laboratory’s expertise—using cryo-EM in Weixlbaumer’s team, while the Berlin group’s Andrea Graziadei utilized in-cell crosslinking mass spectrometry to delve into the processes involved.
The team members, experts in biophysics, including Chauvier and Nils Walter, a professor of chemistry and biophysics at U-M, employed advanced single-molecule fluorescence microscopes to study the dynamic processes of the structure.
“To monitor the speed at which this machinery operates, we tagged each of the two components with different colors,” explained Chauvier. “One fluorescent color was used for the nascent RNA, and another for the ribosome. This method allowed us to separately observe their movements under a high-powered microscope.”
They discovered that the mRNA produced by RNAP was particularly efficiently bound to the small ribosomal subunit (30S) when ribosomal protein bS1 was present, as it aids in the unfolding of the mRNA in preparation for translation within the ribosome.
Furthermore, cryo-EM structures from researchers Webster and Weixlbaumer highlighted an alternate pathway for mRNA delivery to the ribosome, through the engagement of transcription factor NusG or its version RfaH, which threads the mRNA into the entry channel of the ribosome from the opposite side of bS1.
After successfully visualizing the initial phase of coupling RNAP with the ribosome, the research team is eager to continue collaboration to understand how this complex must be rearranged to achieve full functionality.
“This research exemplifies the impact of interdisciplinary collaboration across various countries and oceans,” stated Walter.
Huma Rahil, a doctoral student in Weixlbaumer’s lab, and Michael Webster, who was a postdoctoral fellow in the lab and is now affiliated with The John Innes Centre in the UK, also co-led the publication.