U-M, multinational team of scientists reveal structural link for initiation of protein synthesis in bacteria

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ANN ARBOR—Within a cell, DNA carries the genetic code for building proteins. 

To build proteins, the cell makes a copy of DNA, called mRNA. Then, another molecule called a ribosome reads the mRNA, translating it into protein. But this step has been a visual mystery: scientists previously did not know how the ribosome attaches to and reads mRNA. 

Now, a team of international scientists, including University of Michigan researchers, have used advanced microscopy to image how ribosomes recruit to mRNA while it's being 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 from  Institut de génétique et de biologie moléculaire 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 the book from. How is the book delivered to the reader?"

The researchers discovered that the RNAP transcribing the mRNA deploys two different anchors to rope in the ribosome and ensure a solid footing and start of protein synthesis. This is similar to a foreperson at a construction site overseeing workers installing a complex section of the superstructure, confirming in two redundant ways that all the pieces are fastened securely at critical junctures for maximum stability and functionality.

Understanding these fundamental processes holds great potential for developing new antibiotics that target these specific pathways in bacterial protein synthesis, according to the researchers. 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 bacteria by cutting off multiple pathways.  

"We know there is an interaction between the RNAP, the ribosome, transcription factors, proteins and mRNA," said U-M senior scientist Adrien Chauvier, one of four co-leaders of the study. "We could target this interface, specifically between the RNAP, ribosome, and mRNA, with a compound that interferes with the recruitment or the stability of the complex."

The team developed a mechanistic framework to show how the various components of the complex work together to bring freshly transcribed mRNAs to the ribosome and act 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-billionth 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-off nucleus, where RNAP serves as the "interpreter," breaking down genetic instructions into smaller bites. This dynamo of an enzyme transcribes, or writes, the 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 "roomier" cytoplasm, where it is translated into proteins, the basic building blocks of life. 

In prokaryotes, which lack a distinct nucleus and internal membrane "wall", transcription and translation happen simultaneously and in close proximity to each other, allowing the RNAP and the ribosome to directly coordinate their functions and cooperate with each other.

Bacteria are the best-understood prokaryotes, and because of their simple genetic structure, provided the team with the ideal host to analyze the mechanisms and machinery involved in the ribosome-RNAP coupling during gene expression.

The researchers employed various technologies and methodologies per each lab's specialty—cryo-EM in Weixlbaumer's group, and the Berlin group's in-cell crosslinking mass spectrometry carried out by Andrea Graziadei — to examine the processes involved. 

With expertise in biophysics, Chauvier and Nils Walter, U-M professor of chemistry, biophysics, utilized their advanced single molecule fluorescence microscopes to analyze the kinetics of the structure. 

“In order to track the speed of this machinery at work, we tagged each of the two components with a different color,” Chauvier said. “We used one fluorescent color for the nascent RNA, and another one for the ribosome. This allowed us to view their kinetics separately under the high-powered microscope."

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

The cryo-EM structures of Webster and Weixlbaumer pinpointed an alternative pathway of mRNA delivery to the ribosome, via the tethering of RNA polymerase by the coupling transcription factor NusG, or its paralog, or version, RfaH, which thread the mRNA into the mRNA entry channel of the ribosome from the other side of bS1.

Having successfully visualized the very first stage in establishing the coupling between RNAP and the ribosome, the team looks forward to further collaboration to find out how the complex needs to rearrange to become fully functional.

"This work demonstrates the power of interdisciplinary research carried out across continents and oceans," said Walter.

Huma Rahil, a doctoral student in the Weixlbaumer lab, and Michael Webster, then a postdoctoral fellow in the lab and now of The John Innes Centre in the United Kingdom, co-led the paper as well.

Study: Molecular basis of mRNA delivery to the bacterial ribosome

Written by Paul Avedisian, Center for RNA Biomedicine

 

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