Inside a cell, DNA carries the genetic code to build proteins.
To make proteins, the cell makes a copy of DNA, called mRNA. Then, another molecule called the ribosome reads the mRNA and translates it into protein. But this step has been a visual mystery: until now scientists did not know how the ribosome binds to the mRNA and how it reads.
Now, a team of international scientists, including researchers at the University of Michigan, has used advanced microscopy to image how ribosomes are recruited to mRNA as it is transcribed by an enzyme called RNA polymerase or RNAP. Their results, which examine the process in bacteria, are published in the journal Science.
“Understanding how the ribosome captures or ‘recruits’ the mRNA is a prerequisite for everything that comes after, such as understanding how it can even begin to interpret the information encoded in the mRNA,” said Albert Weixlbaumer, a researcher at the Institut de génétique et de biologie moleculeire et cellulaire in France, who co-led the study. “It’s like a book. Your task is to read and interpret a book, but you don’t know where to get it from. How is the book delivered to the reader?”
The researchers discovered that the RNAP that transcribes the mRNA deploys two different anchors to hold the ribosome and ensure a solid base and the start of protein synthesis. This is similar to a foreman on a construction site supervising workers installing a complex section of superstructure, confirming in two redundant ways that all parts are securely fastened at critical joints for maximum stability and functionality.
According to the researchers, understanding these fundamental processes has great potential for developing new antibiotics that target these specific pathways in bacterial protein synthesis. Traditionally, antibiotics have targeted the ribosome or RNAP, but bacteria often find a way to evolve and mutate to create some resistance to those antibiotics. Armed with their new knowledge, the team hopes to outwit the bacteria by cutting off multiple pathways.
“We know that there is an interaction between RNAP, the ribosome, transcription factors, proteins and mRNA,” said UM senior scientist Adrien Chauvier, one of four co-leaders of the study. “We could target this interface, specifically between the RNAP, the ribosome and the mRNA, with a compound that interferes with the recruitment or stability of the complex.”
The team developed a mechanistic framework to show how the complex’s various components work together to deliver newly transcribed mRNAs to the ribosome and act as bridges between transcription and translation.
“First of all, we wanted to find out how the docking of RNAP and the ribosome is established,” said Weixlbaumer. “Using purified components, we reassembled the complex, 10 billionths of a meter in diameter. We saw them in action using cryo-electron microscopy (cryo-EM) and interpreted what they were doing. We then needed to see if the behavior of our purified components could be recapitulated in different experimental systems.”
In more complex human cells, DNA resides in the walled nucleus, where RNAP acts as an “interpreter,” breaking down genetic instructions into smaller fragments. This dynamo of an enzyme transcribes, or writes, DNA into mRNA, which represents a specifically selected copy of a small fraction of the genetic code that is moved to the ribosome in the much “more spacious” cytoplasm, where it is translated into proteins, the basic elements of life.
In prokaryotes, which lack a distinct nucleus and inner membrane “wall,” transcription and translation occur simultaneously and in close proximity to each other, allowing RNAP and the ribosome to directly coordinate their functions and cooperate with each other.
Bacteria are the best understood prokaryotes and, due to their simple genetic structure, provided the team with the ideal host to analyze the mechanisms and machinery involved in ribosome-RNAP coupling during gene expression.
The researchers used various technologies and methodologies depending on the specialty of each laboratory (cryo-EM in the Weixlbaumer group and intracellular cross-linking mass spectrometry from the Berlin group carried out by Andrea Graziadei) to examine the processes involved.
With a background in biophysics, Chauvier and Nils Walter, a U-M professor of chemistry and biophysics, used their advanced single-molecule fluorescence microscopes to analyze the kinetics of the structure.
“To track the speed of this machinery in operation, we label each of the two components with a different color,” Chauvier said. “We used one fluorescent color for the nascent RNA and another for the ribosome. This allowed us to see their kinetics separately under the high-power microscope.”
They observed that mRNA arising from RNAP bound to the small ribosomal subunit (30S) particularly efficiently when the ribosomal protein bS1 was present, which helps the mRNA unfold in preparation for translation within the ribosome.
Cryo-EM structures by Webster and Weixlbaumer identified an alternative pathway of mRNA delivery to the ribosome, through binding of RNA polymerase by the coupling transcription factor NusG, or its paralog or version, RfaH, which threads the mRNA at the entry of the mRNA. Ribosome channel from the other side of bS1.
Having successfully visualized the first stage in establishing docking between RNAP and the ribosome, the team hopes to continue collaborating to discover how the complex needs to reorganize to become fully functional.
“This work demonstrates the power of interdisciplinary research conducted across continents and oceans,” Walter said.
Huma Rahil, a PhD student in the Weixlbaumer lab, and Michael Webster, then a postdoctoral fellow in the lab and now at the John Innes Center in the UK, also co-led the paper.