Rubisco function, evolution, and engineering

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin Benson Bassham (CBB) cycle is the dominant carbon fixation pathway on earth, driving >99.5% of the ~120 billion tons of carbon that are "fixed" as sugar, by plants, algae and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast and it is a bottleneck in flux through the pathway. This presents a paradox - why hasn't rubisco evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs, and that rubisco variants have been optimized for their native physiological environment. Here we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and, thereby, promote plant growth

Towards Self-Replicating Informational Polymers

The capability to transmit information from generation to generation is an essential feature of life. In all terrestrial life, DNA and RNA contain information in the form of a sequence of monomers and are copied in every generation. The RNA world hypothesis posits that there was a time in the history of life when all cellular functions were accomplished by RNA catalysts. However, the initial emergence of those RNA catalysts is not yet fully understood. Nonenzymatic RNA polymerization has been proposed as a potential stepping- stone from prebiotic chemistry to the RNA world. In searching for alternatives to the chemically trapped triphosphate nucleotides found in modern biology, chemical modifications of RNA have been discovered that allow for the copying of RNA. However, the copying of sequences rich in adenine and uracil residues remains a significant challenge. In chapter 2 we use chemically activated oligonucleotides as catalysts to copy all four monomers sequentially, potentially creating a route for the copying of any sequence without a polymerase and contributing to a model for the emergence of evolution. Replacing uracil with 2-thiouracil and 2-thiothymine, modified forms of uracil found in modern life, has proven to improve the reactivity and fidelity of nonenzymatic RNA polymerization. In chapter 3, we tested these alternatives to uracil as substrates and components of an RNA polymerase ribozyme. We discovered that they were superior in the context of ribozyme mediated RNA polymerization both in terms of faster rate and higher fidelity. We then synthesized ribozymes in which every instance of uracil was replaced by either 2-thiouracil or 2-thiothymine and found them to retain some activity. We hypothesize that these alternative nucleobases could have conferred significant benefits to early life forms. To explore alternative genetic systems to DNA and RNA, in chapter 4 of this thesis we synthesized an organic-soluble copolymer with two different monomers capable of reversible covalent bond formation with one another. We show that information copying is possible with this polymer by synthesizing a polymer with a sequence complementary to a template while it is still covalently bound to that template, demonstrating that nucleic acids are not the only molecules capable of information storage and replication. Together, these results assist in the construction of artificial life and expand the possibilities for the emergence of life.Chemistry and Chemical Biolog

Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides

The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world. DOI: http://dx.doi.org/10.7554/eLife.17756.00

A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases_NGS data analysis

<p>These data files are associated with the NGS analysis done in the publication :"A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases". This compressed file contains the raw file as well as the processed files to arrive at the conclusions published. The python scripts used for processing the data are available on github (link provided in the manuscript).</p&gt

Translation factors direct intrinsic ribosome dynamics during translation termination and ribosome recycling.

Characterizing the structural dynamics of the translating ribosome remains a major goal in the study of protein synthesis. Deacylation of peptidyl-tRNA during translation elongation triggers fluctuations of the pretranslocation ribosomal complex between two global conformational states. Elongation factor G-mediated control of the resulting dynamic conformational equilibrium helps to coordinate ribosome and tRNA movements during elongation and is thus a crucial mechanistic feature of translation. Beyond elongation, deacylation of peptidyl-tRNA also occurs during translation termination, and this deacylated tRNA persists during ribosome recycling. Here we report that specific regulation of the analogous conformational equilibrium by translation release and ribosome recycling factors has a critical role in the termination and recycling mechanisms. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis. Ribosome, tRNA and translation factor structural rearrangements are hypothesized to have important mechanistic roles throughout protein synthesis. Some of the most well-characterized conformational changes of the translational machinery include the movements of tRNAs from their classical to their hybrid ribosome binding configurations, movement of the ribosomal L1 stalk from an open to a closed conformation, and the counterclockwise rotation, or ratcheting, of the small (30S) ribosomal subunit relative to the large (50S) subunit The essential features of our dynamic model have recently been largely validated. smFRET studies of pretranslocation complexes have reported spontaneous and reversible intersubunit rotation between two major conformations, nonratcheted and ratcheted 10 , as well as fluctuations of the L1 stalk between open and closed conformations 11 (J.F and R.L.G., unpublished data). Collectively, these studies revealed that the equilibrium constants governing the nonratcheted#ratcheted ribosome and open#closed L1 stalk equilibria are closely correlated 11 , reinforcing the idea that these dynamic processes are coupled. Consistent with this model, two recent cryo-EM studies used classification methods to reveal the existence of both GS1-and GS2-like conformations within a single pretranslocation sample 6,7 , without any detectable intermediates. Nevertheless, our GS1#GS2 model certainly does not incorporate all of the dynamic complexity encompassed by a B2.5-MDa biomolecular machine. In addition, it remains entirely possible that short-lived and/or rarely sampled intermediates have so far eluded detection by smFRET experiments and cryo-EM reconstructions. Thus, the GS1#GS2 model represents the simplest dynamic model that is consistent with the available data, providing a convenient framework for investigating the dynamics of the translating ribosome. We have previously reported that reversible transitions between GS1 and GS2 are prompted by peptidyltransfer to either an A-site aminoacyl-tRNA (aa-tRNA) or to the antibiotic puromycin 8 . Puromycin mimics the 3¢-terminal residue of aa-tRNA 12 but, unlike a fully intact aa-tRNA, dissociates rapidly from the A site upon peptidyltransfer. Therefore, deacylation of P-site peptidyl-tRNA alone, regardless of A-site occupancy, is necessary and sufficient to trigger GS1#GS2 fluctuations. Binding of the GTPase ribosomal translocase, elongation factor G (EF-G), stabilizes GS2 (refs. 8, Beyond elongation, a deacylated tRNA also occupies the P site during translation termination and ribosome recycling, raising the possibility that regulation of the GS1#GS2 equilibrium may be mechanistically important throughout these additional stages of protein synthesis. During termination, a stop codon in the A site o

Experimental and Computational Evidence for a Loose Transition State in Phosphoroimidazolide Hydrolysis

Phosphoroimidazolides play a critical role in several enzymatic phosphoryl transfer reactions and have been studied extensively as activated monomers for nonenzymatic nucleic acid replication, but the detailed mechanisms of these phosphoryl transfer reactions remain elusive. Some aspects of the mechanism can be deduced by studying the hydrolysis reaction, a simpler system that is amenable to a thorough mechanistic treatment. Here we characterize the transition state of phosphoro­imid­azolide hydrolysis by kinetic isotope effect (KIE) and linear free energy relationship (LFER) measurements, and theoretical calculations. The KIE and LFER observations are best explained by calculated loose transition structures with extensive scissile bond cleavage. These three-dimensional models of the transition state provide the basis for future mechanistic investigations of phosphoro­imid­azolide reactions

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth

Protein design using structure-based residue preferences.

Recent developments in protein design rely on large neural networks with up to 100s of millions of parameters, yet it is unclear which residue dependencies are critical for determining protein function. Here, we show that amino acid preferences at individual residues-without accounting for mutation interactions-explain much and sometimes virtually all of the combinatorial mutation effects across 8 datasets (R2 ~ 78-98%). Hence, few observations (~100 times the number of mutated residues) enable accurate prediction of held-out variant effects (Pearson r > 0.80). We hypothesized that the local structural contexts around a residue could be sufficient to predict mutation preferences, and develop an unsupervised approach termed CoVES (Combinatorial Variant Effects from Structure). Our results suggest that CoVES outperforms not just model-free methods but also similarly to complex models for creating functional and diverse protein variants. CoVES offers an effective alternative to complicated models for identifying functional protein mutations