Within each cell, DNA holds the instructions for building the proteins essential to life. To carry out this task, cells first create a copy of the DNA called messenger RNA (mRNA). The mRNA is then translated into protein by ribosomes, which read the sequence of nucleotides in the mRNA. However, until recently, the precise process by which ribosomes bind to and begin reading mRNA during protein synthesis was not fully understood.
Now, an international team of scientists, including researchers from the University of Michigan, has uncovered critical insights into how ribosomes recruit mRNA while it is being transcribed by an enzyme called RNA polymerase (RNAP). Their findings, published in the journal Science, provide a clearer understanding of the early stages of protein synthesis in bacteria, which could have implications for new antibiotic development.
The discovery centers on how RNAP, which is responsible for transcribing DNA into mRNA, coordinates with the ribosome to ensure that mRNA is efficiently recruited for translation. “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, co-lead researcher from the Institut de génétique et de biologie moléculaire et cellulaire in France.
This research is crucial because it addresses a previously elusive aspect of protein synthesis. It’s analogous to reading a book: if you don’t know how to get the book to the reader, the reading process can’t begin. This study reveals how RNAP acts as a guide, ensuring that the ribosome properly attaches to the mRNA.
Mechanism of Ribosome Recruitment
The team discovered that RNAP employs two separate strategies to anchor the ribosome and guide it to the mRNA, ensuring proper positioning and the start of protein synthesis. This process is likened to a foreperson overseeing workers at a construction site, making sure all components are securely fastened before proceeding. These redundant mechanisms provide stability, ensuring the ribosome is precisely aligned to begin translating the mRNA.
The ability to understand this interaction could revolutionize the development of antibiotics. Most antibiotics work by targeting either the ribosome or RNAP to inhibit protein synthesis in bacteria. However, bacterial resistance to antibiotics has been a growing problem, as bacteria evolve mechanisms to evade these drugs. With this new knowledge, scientists hope to target the interface between RNAP, the ribosome, and mRNA—disrupting multiple steps in the process and making it harder for bacteria to develop resistance.
“We know there is an interaction between the RNAP, the ribosome, transcription factors, proteins, and mRNA,” said Adrien Chauvier, senior scientist at the University of Michigan and one of the co-leaders of the study. “We could target this interface with a compound that interferes with the recruitment or the stability of the complex.”
Cryo-EM and Single-Molecule Techniques
The research team used cutting-edge technologies to unravel the complex process. They employed cryo-electron microscopy (cryo-EM), a powerful imaging technique that allows scientists to visualize molecular structures at near-atomic resolution. With cryo-EM, the researchers were able to observe how RNAP and ribosomes interact during transcription and translation.
Additionally, the team used single-molecule fluorescence microscopy to track the movement of RNAP and ribosomes in real-time. By tagging the mRNA and ribosome with different fluorescent markers, they were able to observe how the two components interacted and how the ribosome efficiently bound to the emerging mRNA.
The results revealed that the ribosome binds to the nascent mRNA as it is transcribed by RNAP, with particular efficiency when the ribosomal protein bS1 is present. This protein helps unfold the mRNA, preparing it for translation within the ribosome. Furthermore, the cryo-EM structures identified an alternative pathway by which mRNA can be delivered to the ribosome through the action of the transcription factor NusG or its paralog, RfaH. These factors tether RNAP to the ribosome, allowing mRNA to be threaded into the ribosomal entry channel from the other side of bS1.
Transcription and Translation in Prokaryotes
The study was conducted in bacteria, which are prokaryotic cells. Prokaryotes lack a defined nucleus, and as a result, transcription and translation occur simultaneously and in close proximity within the cell. This direct coordination between RNAP and the ribosome allowed the researchers to study the coupling of these two processes in real-time. In more complex eukaryotic cells, such as human cells, transcription takes place in the nucleus, and mRNA must be transported to the cytoplasm for translation, making the process more compartmentalized.
Because bacteria provide a simple and accessible model system, they were the ideal organisms for the study of ribosome recruitment. In bacteria, RNAP is responsible for transcribing DNA into mRNA, and the ribosome quickly binds to the mRNA to begin translating it into proteins. This study in bacteria offers crucial insights into the basic mechanisms that also apply to eukaryotic cells, despite the differences in cellular architecture.
Implications for Antibiotic Development
The researchers believe their findings could have a significant impact on the development of new antibiotics. By targeting the interface between RNAP, the ribosome, and mRNA, scientists could potentially develop drugs that disrupt multiple points in the protein synthesis pathway, making it more difficult for bacteria to evolve resistance. Currently, most antibiotics work by targeting either the ribosome or RNAP, but resistance to these drugs is becoming increasingly common. By understanding the finer details of how these molecular machines cooperate, new therapeutic strategies can be designed.
“We’ve gained a better understanding of the steps involved in recruiting the ribosome to mRNA, which can be exploited in developing more effective antibiotics,” said Weixlbaumer. “If we target the steps where the mRNA is captured, we could potentially block protein synthesis in bacteria in new and more efficient ways.”
Collaborative Research Across Disciplines
This work also highlights the power of interdisciplinary collaboration in scientific research. The team combined expertise in biophysics, biochemistry, and structural biology, using a variety of tools and technologies to approach the problem from different angles. From cryo-EM and single-molecule fluorescence microscopy to mass spectrometry and computational modeling, each technique contributed to the overall understanding of ribosome recruitment and translation initiation.
“This work demonstrates the power of interdisciplinary research carried out across continents and oceans,” said Nils Walter, a professor at the University of Michigan who co-led the study. “By combining our collective knowledge and advanced technologies, we’ve uncovered new insights into one of biology’s most fundamental processes.”
As the team continues to explore how these molecular machines function together, their research promises to advance our understanding of cellular processes and provide new avenues for drug development. With bacterial resistance to existing antibiotics on the rise, this work may offer a new path forward in the fight against infectious diseases.
Source: University of Michigan