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Four billion years ago, a single-celled organism floated in the primordial soups of early earth. This ancient organism already had many of the makings of modern life forms, including a functional ribosome, that nanoscopic machine that makes the thousands of unique proteins that cells require for survival.
Two separate ribosomal subunits, the large and the small, come together to decode mRNA messages and build proteins. The catalytic activity of the ribosome is derived from ribosomal RNA that resides in each of the subunits — in the large subunit, a strand of RNA catalyzes the peptide bonds between amino acids, while the small subunit contains an RNA that decodes the precise position in the mRNA message to begin translation. The process of translation is such a delicate dance that it took hundreds of scientists several decades to decipher.
Now, scientists want to modify the ribosome, expanding its capabilities to synthesize not just proteins, but also other types of polymers. This effort has been hindered, in part, because of the conserved nature of this molecular machine — even a minor modification to its structure could prove disastrous and kill off an unlucky organism in the process. In 2015, a team of researchers at Northwestern University created an ‘orthogonal’ ribosome by tethering the large and small subunits together. The new machine, called Ribo-T, was reported in Nature to considerable fanfare. It marked the first time that cells were able to grow, albeit slowly, in the absence of wild-type ribosomes (see Erik Carlson’s “behind-the-scenes” look at Ribo-T in this ACS Synthetic Biology article). Now, that same team has improved upon their designs, creating a Ribo-T v2.0 that could enable researchers to engineer cells to produce incredible new polymers – not just proteins.
I sat down with Dr. Erik Carlson, a joint first author on this study (together with Anne d’Aquino, a graduate student in the Jewett lab), to learn more about the new version of Ribo-T, discuss experimental challenges, and highlight future plans.
This interview with Dr. Carlson on “Engineered ribosomes with tethered subunits for expanding biological function”, published in Nature Communications (an open access journal), has been edited for clarity.
Niko McCarty: What was the original motivation for making a tethered ribosome in the 2015 Nature paper?
Dr. Erik Carlson: Well, the ribosome is a remarkably complex machine that can do some really cool things, including template-based assembly of polymers. That ability alone makes it interesting. Unfortunately, it is very challenging to use the ribosome as a tool to make polymers with more than the 20 or so amino acids that cells use in nature. In that regard, the ribosome can only access a very limited chemical space. In all kingdoms of life, the ribosome has two separate subunits, each with very conserved and distinct functions. The small subunit is involved in mRNA recognition, while the large subunit is really the catalytic core; this is where peptide bonds are formed. This catalytic activity is executed by a large chunk of RNA in the large subunit, called the 23S rRNA. The problem with trying to engineer the ribosome is that, if you mess with it too much, the cell will die. The ribosome is an essential machine for life; it is difficult to move its function away from those 20 fundamental amino acids. Our motivation for creating Ribo-T was, therefore, to physically link the large subunit to a functionally orthogonal small subunit, enabling us to begin engineering the catalytic core and expand its repertoire of polymers without killing cells in the process!
Niko: Why is it preferable to engineer the large ribosomal subunit, rather than use genomic recoding, to expand the chemical space available to cells?
Erik: Well, genomic recoding has been incredibly useful for adding non-canonical amino acids to proteins, but you’re still fundamentally limited to that amino acid space. Non-canonical amino acids are, in many respects, the same as natural amino acids; they have the same backbone structure with a slightly different R group. If you want to go beyond that quite limited sequence space and begin to use the ribosome as a template-directed polymer building machine for, say, ester bond formation, you have to engineer the ribosome itself.
Niko: After developing the original Ribo-T, why did you decide to return to this project and engineer a second version, reported in this 2019 Nature Communications article?
Erik: It was a very long process to engineer the original Ribo-T; it took us more than five years to create that orthogonal ribosome. When we finally transplanted it into E. coli lacking the wildtype ribosomal RNA, it resulted in cells that were quite sick, with doubling times a bit less than 2 hours. This slow growth made the Ribo-T strains, from a practical standpoint, very difficult to work with.
The main motivation for this new paper was to make Ribo-T a much more robust and workable strain for everyone to use. We really want Ribo-T to serve as a tool for people to study the ribosome. In this Nature Communications paper, we therefore place a lot of emphasis on the improved growth rates, strain behavior, and engineering potential of this v2.0 Ribo-T.
Niko: What specific improvements did you make to Ribo-T to improve cellular growth rate?
Erik: We made two major improvements. First, we upgraded the position, length and sequence of the RNA tether that physically connects the small subunit to the large subunit. This tether connects helix 101 in the large subunit with helix 44 in the small subunit. In the original paper, we just used a poly-A sequence for this tether, but perhaps this was negatively impacting growth rate. Also, we optimized the physical length of the tether. In the end, we identified many tethers that resulted in improved translation and growth rates. The second major improvement relates to the orthogonal pair itself; prokaryotic ribosomes contain something called an anti-Shine-Dalgarno sequence, which recognizes a corresponding Shine-Dalgarno sequence on mRNAs. This pair is what enables initiation of translation. In our original paper, we simply used Shine-Dalgarno / anti-Shine-Dalgarno pairs developed for untethered ribosomes by Jason Chin’s lab in Cambridge. In this new paper, we selected pairs in the tethered context that enhance Ribo-T function.
Niko: Tell me about your background in chemical engineering; how did that alter your strategy to engineer Ribo-T?
Erik: I completed my undergraduate degree in chemical engineering, but my research interests have always been rooted in biology. The beauty of chemical engineering is that it really trains you to think about a process at every level, from a molecular to global scale. It is because of my background in chemical engineering that I often think of the interactions between many levels of complexity in cells, ranging from atoms to macromolecular structures. When I decided to do a PhD, I remember interviewing at Northwestern and meeting Mike Jewett. After speaking with him, I instantly became hooked on the Ribo-T idea. When I started working on the project, I realized early on that my real passion is in engineering approaches to fundamental biology. It took decades to uncover the structure and mechanisms of the ribosome; moving that work into an application space really brought out the best science in me.
Niko: This paper had joint first authors – you and Anne d’Aquino. What was it like to work together?
Erik: Anne is an incredible researcher, who made the tether libraries, ran the selections and characterized the winners. It was amazing to work together, and the product benefited as a result. Working with friends always makes the job easier. This project, as a whole, brought together a lot of scientists and engineers with varied backgrounds. In an ambitious project like this, you really can’t be expected to have expertise in everything, and so it’s crucial that you find collaborations, seek out help, and share responsibilities among authors.
Niko: What were some of the major challenges that you encountered during this project?
Erik: In the original Ribo-T paper, we were basically just taking “shots in the dark” to figure out where we could connect the tether between the subunits. For this revamped project, we did a much deeper characterization of tethers, screening through large libraries of variants to determine optimal sequences and lengths. Other than that, this paper was a bit “cut and dry”. We already knew the system quite well, and had a good sense of the improvements that we wanted to make.
Niko: What comes next for you?
Erik: Well, I certainly love the ribosome, but I decided to transition to plant synthetic biology for my postdoctoral research. I wanted to work on something totally different, so I joined Elizabeth Sattely’s lab at Stanford University. The resources here are terrific, I enjoy the day-to-day research in academia, and I like that I don’t have the added baggage of writing a thesis, serving as a TA, or other PhD-student responsibilities. For the moment, I am just enjoying my research – whatever comes next will sort itself out.
Dr. Erik Carlson is a postdoctoral fellow at Stanford University, where he works in the Sattely lab in the Chemical Engineering department. He previously completed his PhD in Chemical and Biological Engineering at Northwestern University, under the guidance of Michael Jewett.
Anne d’Aquino is joint first author on this 2019 Nature Communications article. d’Aquino is a graduate student in Michael Jewett’s lab at Northwestern and an NSF Graduate Research Fellow.