Understanding the last step of protein synthesis – the basic process of translating DNA into its final protein product – just became more clear both literally and figuratively. This final phase, called recycling, is essential for the proper function of all cells.
Using a three-dimensional cryo-electron microscope to directly observe protein structure, investigators at the University of Pennsylvania School of Medicine and the State University of New York, Albany can now visualize the exact configuration of a molecule called ribosome recycling factor (RRF) in the common bacteria Escherichia coli. This may help guide the design of new antibiotics aimed at inhibiting RRF-related steps of protein synthesis.
“Every living organism has to have this last step, the recycling of spent protein synthesis machinery for the next round of translation,” says Akira Kaji, PhD, Professor of Microbiology at Penn. “Strangely, at this day and age, this most fundamental process remained vague until we launched our studies of RRF.”
Most antibiotics influencing protein synthesis act by stopping its molecular machinery. However, none as yet target the recycling step. “We believe RRF is one of the best candidates for a new antibiotics target because the mechanism involved in recycling of the protein-making machinery is different in eukaryotes versus prokaryotes, that is humans versus bacteria,” says Kaji. “With the emergence of antibiotic-resistant pathogens, this will be the best avenue of devising new antibiotics.”
Thirty Years of Searching
The ribosome is the structure within cells on which amino acids are strung together to make proteins with the aid of transfer RNA (tRNA) and messenger RNA (mRNA). Kaji has spent the past 30 years working out the last step of protein synthesis. RRF, in conjunction with elongation factor G (EF-G), moves along the ribosome removing mRNA and tRNA, readying it to make more proteins. In this latest chapter, Kaji and colleagues report the three-dimensional image of RRF bound to the E. coli ribosome.
In an earlier paper by Kaji and colleagues from Sweden, the crystal structure of RRF showed that RRF mimics the L-shape and dimension of tRNA. Chemical probing by Kaji and colleagues at the University of California, Santa Cruz showed the approximate ribosomal binding site of RRF. In the current PNAS paper, direct observation of the RRF-ribosome structure revealed the exact ribosomal position of bound RRF. It further showed that part of the ribosome contorts by a significant amount – molecularly speaking – when RRF binds to it.
More precisely, the position of the key helices of the ribosomal small and large subunits that hold mRNA move inward, suggesting that this movement may be essential for the release of mRNA from the ribosome. In addition, the RRF binding sites are very close to where the two ribosomal subunits are held together, which explains an earlier observation that the disassembly reaction by RRF may be followed by dissociation of the two subunits.
In short, the recycling process goes like this: RRF, along with EF-G, binds to the ribosome. This promotes the release of tRNAs by the movement of RRF, similar to tRNA movement. “This is the first example of a functional mimic of tRNA by a protein,” adds Kaji. After the tRNAs leave, RRF, EF-G, and mRNA also detach from the ribosome. The released ribosome is now empty and free to start a new session of translating mRNA into protein. Where RRF binds is near the key ribosomal spot holding mRNA. “Since the main function of RRF is to release mRNA, this makes sense in terms of function,” explains Kaji.
Humans have an RRF analogue in the mitochondria, the respiratory organelle within cells. “One may argue that proposed antibiotics against RRF may influence mitochondrial protein synthesis,” notes Kaji. However, commonly used antibiotics such as erythromycin and tetracycline kill bacteria but are virtually harmless to humans, showing little side effect despite their influence on mitochondrial protein synthesis. “With rational drug design it is even possible to design anti-RRF which would only influence bacterial RRF,” says Kaji.
His lab is currently identifying the ribosomal site to which RRF is moved from the currently identified position. “It is from this position where RRF performs the final and the most important act – release of mRNA,” says Kaji. “The fourth step of protein synthesis within human cells is shrouded in complete mystery and nothing is known. This fundamental step must be elucidated before we can take advantage of the fact that the same step is catalyzed by RRF in bacteria.”
Other scientists contributing to this work are: Rajendra K. Agrawal, Manjuli R. Sharma, and Timothy M. Booth from the New York State Department of Health; Michael C. Kiel and Go Hirokawa from Penn; and Christian M.T. Spahn, Robert A. Grassucci, and Joachim Frank from the Howard Hughes Medical Institute. Agrawal and Frank are also affiliated with the State University of New York, Albany. This research was funded in part by the National Institutes of Health and the National Science Foundation.