Ribosomal ties that bind

The ribosome is the cellular complex of proteins and RNA molecules that synthesizes proteins. An artificial ribosome in which the two main subunits are tethered together creates opportunities for engineering this process. See Letter p.119

To engineer a system is to demonstrate a mastery of physical understanding. Mechanical engineers harness a deep understanding of fundamental physics to design new motors. Similarly, biologists are using the current explosion in information about molecular structure and function to engineer biological systems. The ribosome — the macromolecular complex containing RNAs and proteins that translates the genetic code — represents one of nature's most sophisticated machines. Engineering ribosomes would enable experimental manipulation of protein synthesis and provide deeper insights into cellular and molecular biology. On page 119 of this issue, Orelle et al.¹ describe drastic, but simple, engineering of functional ribosomes, in which two separate subunits are linked as one.

Ribosomes in all organisms exist as two separate subunits, large and small, each consisting of one or two RNA chains and scores of proteins (Fig. 1). The small subunit is where messenger RNA and transfer RNA (tRNA) molecules interact to read the genetic code, and the large subunit is where the growing protein chain is synthesized from amino acids attached to tRNAs. In prokaryotic organisms (those lacking a nucleus, such as bacteria), these are the 30S and 50S subunits, respectively. The two subunits cooperate to control the precise movements required to make a protein chain.

Figure 1: A tethered ribosome.

a, The complex of proteins and RNA molecules known as the ribosome carries out the process of protein translation, in which an amino-acid chain is built according to a messenger RNA sequence. Translation is initiated when the ribosome's small subunit and a transfer RNA (tRNA) molecule attach to a 'start codon' (nucleotide sequence AUG) on an mRNA. A large ribosomal subunit then joins the assembly to create a full ribosome that begins translation. b, Orelle et al.¹ have generated a ribosome in which the small and large subunits are attached by a short length of RNA. Their tethered ribosome is able to carry out normal translation.

Protein translation is initiated through a complex process by which a 30S subunit binds to an mRNA and, together with a special initiator tRNA, finds the three-nucleotide sequence (codon) in the mRNA that indicates where translation should start. A 50S subunit then joins the assembly to create an intact 70S ribosome that is competent to translate. Structural biology using X-ray diffraction and electron microscopy has revealed the detailed and global features of the ribosomal subunits and the fully assembled 70S ribosomal particle², and biophysical and mechanistic studies have sketched the time course of this process^3,4. However, a fundamental challenge to engineering ribosomes is that they come in two pieces that must function as one.

Biochemists have been engineering biomolecules for decades — proteins with new folds and functions, and nucleic acids that control gene expression or form complex architectures. A goal of such synthetic biology is to engineer biomolecules to perform functions that are independent of their normal role in the cell, for example a separate pool of ribosomes that can translate using a distinct genetic code⁵. As a step towards this goal, specialized bacterial ribosomes were created⁶ that have a mutated sequence in the ribosomal RNA that normally binds to a specific mRNA sequence to position the 30S subunit for initiation. These ribosomes can be guided to translate only mRNAs with certain sequences. Extension of this technology would allow the engineering of ribosomes that perform tasks that are independent of the normal 'worker-bee' ribosomes. However, in this scenario, a normal 50S subunit would interact with both the mutant and the normal 30S subunits, so two truly independent populations of ribosomes are not possible.

In a deft design, Orelle et al. circumvent this problem by connecting the two subunits using a tether made of RNA. Their approach was guided by the structure of the ribosome, in which two RNA hairpins (structures in which the RNA folds back on itself) on the surface of the 30S and 50S subunits are located in close proximity (less than 50 ångströms apart). Both hairpins had been altered drastically in earlier studies without affecting ribosomal function^7,8. The authors designed a ribosomal RNA construct that stitched together the 16S and 23S RNAs (components of the 30S and 50S subunits, respectively) at these hairpins into a single covalent RNA chain. The linker, also made out of RNA, was long enough to provide a flexible tether, because the two subunits move with respect to each other during translation. The authors then expressed this construct in bacteria and harnessed the power of natural selection over multiple generations to find the optimal design for their tethered single-particle ribosome, which they call Ribo-T.

The first obvious question was whether this linked ribosome was functional. Through an array of experiments, the authors demonstrated unambiguously that the Ribo-T translates normally. Working in bacteria in which the normal ribosomal RNA genes had been removed, they showed that Ribo-T can act as the only ribosome type in the cell, supporting normal bacterial growth. Biochemical assays confirmed that Ribo-T is fully active in protein synthesis. The authors then showed the utility of Ribo-T by introducing simultaneous mutations in the tethered ribosome's large subunit to allow elongation of peptide sequences that normally cause stalling of translation, and in its small subunit to confer resistance to an antibiotic. The resulting mutant Ribo-T could elongate the stalling sequence only in the presence of the drug. Thus, the Ribo-T system allows linked changes to both ribosomal subunits.

The results presented by Orelle et al. may be harnessed to allow robust engineering of translation systems. Synthetic biologists have previously used altered tRNAs and mRNAs, or unusual amino acids, to recode existing or new codons, allowing the synthesis of proteins containing unnatural amino acids or other chemical building blocks⁵. With Ribo-T, the centrepiece of translation can be engineered to facilitate new applications for translation. Such engineering should always be done with a deep understanding of the mechanistic basis of protein synthesis.

Perhaps even more important than this potential application in synthetic biology are the crucial questions that Ribo-T raises about our view of translation — one that is formed by the textbook concept of separated ribosomal subunits. How does the tethered ribosome initiate translation on an mRNA as a single entity — is it like a clamshell grabbing onto the mRNA strand? Although normal translation occurs unhindered with Ribo-T, does it perhaps block unusual events such as frameshifting (when the ribosome shifts its reading of a codon sequence owing to a nucleotide insertion or deletion), which may require dynamic flexibility that might be suppressed by the tether? How do these ribosomes terminate translation, and how are they pried off the mRNA to be recycled? The answers to these questions will illuminate the basic mechanisms of translation and will probably cause surprise. Armed with this knowledge, thoughtful engineers will continue to tinker with the ribosome. ¹