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.

“We saw that transgender men treated with testosterone increased their muscle volume by an average of 21 percent over six years, but also that the amount of abdominal fat increased by 70 percent,” says Tommy Lundberg, docent at the Department of Laboratory Medicine, Karolinska Institutet. “In addition, they had more liver fat and higher levels of ‘bad’ LDL cholesterol, which may increase the risk of cardiovascular disease.”

The researchers followed 17 adult transgender men and 16 transgender women who were prescribed treatment with testosterone and oestrogen, respectively. They used magnetic resonance imaging (MRI) to map body composition and measured metabolic risk factors via blood tests, blood pressure and vascular stiffness. The scans were conducted before the start of hormone therapy, after one year and after five to six years.

The results show that long-term hormone therapy leads to several major changes in both body composition and metabolic risk factors, particularly in transgender men. The changes in fat volumes continued over time, while the greatest changes in muscle mass and strength occurred after just one year of treatment.

“Previous studies in this area have been relatively short-term, up to two years,” explains Tommy Lundberg. “Our results show that it is important to continue monitoring the long-term health effects of hormone therapy in transgender individuals to prevent cardiovascular disease and other health issues.”

In transgender women receiving oestrogen treatment, the changes were not as pronounced. Their muscle volume decreased by an average of seven percent after five years of treatment, whereas muscle strength remained unchanged. The transgender women increased their total fat volume but gained less abdominal fat.

Tissue samples from muscle, fat and skin were also taken as part of the study. The next step is to analyse these tissue samples to understand the interaction between genetic sex and sex hormones. The researchers are investigating, among other things, how hormone treatment affects skeletal muscle gene expression and the mechanisms behind changes in adipose tissue.

“In addition to the health aspects, our research contributes to increased knowledge about reasonable expectations of the masculinising and feminising effects of sex hormone treatment,” says Tommy Lundberg. “However, some of the changes were relatively modest and should raise caution regarding expectations of long-term and large changes in this patient group.”

The research was funded by Region Stockholm, the Thuring Foundation, the 1.6 Million Club, the Centre for Innovative Medicine at Karolinska Institutet, the Swedish Research Council, the Swedish Medical Association, the Novo Nordisk Foundation and the European Foundation for Studies of Diabetes.

Two of the co-authors are employed by AMRA Medical AB. Tommy Lundberg has been compensated for expert opinions on aspects related to skeletal muscle changes in transgender individuals and reimbursed for travelling to give lectures on the same topic.

In recent years, TNT has started to be replaced with DNAN, but until now very little was known about how this substance impacts the environment and how long it can remain in the soil.

Researchers at the University of York have been studying the environmental impact of the explosive, TNT, for more than a decade. They have shown that the chemical compound, which is used by the military around the world, remains in the roots of plants where it inhibits growth and development.

Now a new study, led by Professor Neil Bruce at the University of York’s Department of Biology and Director of the Centre for Novel Agricultural Products (CNAP), however, has shown that DNAN has similar effects to TNT, but accumulates throughout the plant and lingers for longer.

Professor Neil Bruce said: “Similarly to TNT, DNAN reacts with a key plant enzyme, generating reactive superoxide, which is highly damaging to cells. Over the course of our research we have genetically engineered plants to successfully detoxify land contaminated with munitions.

“Unfortunately DNAN is a very different story to TNT, as it accumulates in the above ground parts of the plant. While plants can use natural processes to reduce the toxicity of TNT, our studies found that plants appear to have no natural way of fighting off the toxic effects of DNAN, meaning that it persists in the plant and is toxic at much lower concentrations.”

Researchers warn that as DNAN is present throughout the plant and not just the root system, as is the case with TNT, there is a greater risk of animals eating the infected plant, introducing the toxin into the food chain.

In previous studies by the York team, genetically modified grass was grown on land contaminated with military explosives, which successfully degraded contaminants to non-detectable levels in their plant tissues, but as yet there is currently no such method to remove or reduce DNAN.

The US is estimated to have over 10 million hectares of military land contaminated with constituents of explosives and the US government estimates that remediation of unexploded ordinances on US military training ranges alone will cost $16-165 billion.

Dr Liz Rylott, co-author of the study from the University of York’s Department of Biology, said: “Recent years have seen an escalation in military explosives due to global conflicts, and so we are potentially looking at vast scales of pollution, which means there is an urgent need, and interest in, developing sustainable plant-based remediation strategies.

“We also don’t know what the limits of DNAN toxicity are in humans, so our hope is that our latest research will highlight that more work is urgently needed to understand its effects.”

This research, published in the journal Nature Plants, was funded by the Strategic Environmental Research and Development Program (SERDP) of the U.S. Department of Defense and was in collaboration with researchers at the U.S. Army Engineer Research and Development Center (ERDC), U.S. Army Corps of Engineers.