In a cell, DNA carries the genetic code necessary to build proteins.

To build proteins, the cell makes a copy of DNA, called mRNA. Then another molecule called a ribosome reads the mRNA and translates it into protein. But this step remains a visual mystery: Scientists previously didn’t know how the ribosome attaches to and reads mRNA.

Now, a team of international scientists, including researchers from the University of Michigan, have used advanced microscopy to visualize how ribosomes recruit 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’ mRNA is a prerequisite for everything that comes afterward, such as understanding how it can even begin to interpret the information encoded in mRNA,” said Albert Weixlbaumer, a researcher at the Institute of Genetics and Genetics. of molecular and cellular biology 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 found that the RNAP transcribing the mRNA deploys two different anchors to attach to the ribosome and ensure a strong base and 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 attached at critical points for maximum stability and functionality.

Understanding these fundamental processes holds great potential for developing new antibiotics targeting these specific pathways of bacterial protein synthesis, according to the researchers. Traditionally, antibiotics target the ribosome or RNAP, but bacteria often find a way to evolve and mutate to create some resistance to these antibiotics. With their new knowledge, the team hopes to thwart the bacteria by cutting off several access routes.

We know that there is an interaction between RNAP, ribosome, transcription factors, proteins and mRNA. We could target this interface, specifically between RNAP, ribosome and mRNA, with a compound that interferes with the recruitment or stability of the complex. »

Adrien Chauvier, senior scientist at UM, one of four co-leaders of the study

The team developed a mechanistic framework to show how the different components of the complex work together to bring freshly transcribed mRNAs to the ribosome and serve as bridges between transcription and translation.

“We wanted to find out how the coupling of RNAP and the ribosome is established in the first place,” Weixlbaumer said. “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 had to see whether the behavior of our purified components could be recapitulated in different experimental systems.

In more complex human cells, DNA resides in the isolated nucleus, where RNAP serves as an “interpreter,” breaking down the genetic instructions into smaller pieces. This enzyme dynamo transcribes, or writes, DNA into mRNA, representing a specifically selected copy of a small fraction of the genetic code that is moved to the ribosome in the much more “roomy” cytoplasm, where it is translated into proteins , the basic elements of life.

In prokaryotes, lacking a distinct nucleus and an internal 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 protein expression. Genoa.

The researchers used various technologies and methodologies depending on the specialty of each laboratory – cryo-EM in Weixlbaumer’s group and cross-linking mass spectrometry in cells from the Berlin group carried out by Andrea Graziadei -; to examine the processes involved.

Drawing on their expertise in biophysics, Chauvier and Nils Walter, professor of chemistry and biophysics at UM, used their advanced single-molecule fluorescence microscopes to analyze the kinetics of the structure.

“In order to track the operating speed of this machine, we marked 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 visualize their kinetics separately under a high-power microscope.”

They observed that mRNA emerging from RNAP was bound to the small (30S) ribosomal subunit particularly efficiently when the ribosomal protein bS1 was present, which helps the mRNA unfold in preparation for translation at the inside the ribosome.

Webster and Weixlbaumer’s cryo-EM structures identified an alternative route of mRNA delivery to the ribosome, via attachment of RNA polymerase by the coupling transcription factor NusG, or its paralogue, or version, RfaH , which threads the mRNA into the mRNA input. ribosome channel on the other side of bS1.

Having successfully visualized the very first step in establishing coupling between RNAP and the ribosome, the team looks forward to continuing the collaboration to discover how the complex must reorganize to become fully functional.

“This work demonstrates the power of interdisciplinary research conducted across continents and oceans,” Walter said.

Huma Rahil, a doctoral student in the Weixlbaumer lab, and Michael Webster, then a postdoctoral researcher in the lab and now at the John Innes Center in the United Kingdom, also co-led the paper.