Jessica C. Bowman

RNA Folding and Catalysis Mediated by Iron (II)

Mg2+ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe2+ in the absence of free oxygen as a replacement for Mg2+ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg2+ in RNA folding and function can indeed be served by Fe2+. The results of quantum mechanical calculations show that the geometry of coordination of Fe2+ by RNA phosphates is similar to that of Mg2+. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4–P6 domain RNA is conserved between complexes with Fe2+ or Mg2+. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg2+, and the hammerhead ribozyme are enhanced in the presence of Fe2+ compared to Mg2+. All chemical footprinting and ribozyme assays in the presence of Fe2+ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe2+. The combined biochemical and paleogeological data are consistent with a role for Fe2+ in an RNA World. RNA and Fe2+ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg2+ alone.

Bidentate RNA-magnesium clamps: On the origin of the special role of magnesium in RNA folding

Magnesium plays a special role in RNA function and folding. Although water is magnesiums most common first-shell ligand, the oxyanions of RNA have significant affinity for magnesium. Here we provide a quantum mechanical description of first-shell RNA-magnesium and DNA-magnesium interactions, demonstrating the unique features that characterize the energetics and geometry of magnesium complexes within large folded RNAs. Our work focuses on bidentate chelation of magnesium by RNA or DNA, where multiple phosphate oxyanions enter the first coordination shell of magnesium. These bidentate RNA clamps of magnesium occur frequently in large RNAs. The results here suggest that magnesium, compared to calcium and sodium, has an enhanced ability to form bidentate clamps with RNA. Bidentate RNA-sodium clamps, in particular, are unstable and spontaneously open. Due to magnesiums size and charge density it binds more intimately than other cations to the oxyanions of RNA, so that magnesium clamps are stabilized not only by electrostatic interactions, but also by charge transfer, polarization, and exchange interactions. These nonelectrostatic components of the binding are quite substantial with the high charge and small interatomic distances within the magnesium complexes, but are less pronounced for calcium due to its larger size, and for sodium due to its smaller charge. Additionally, bidentate RNA clamps of magnesium are more stable than those with DNA. The source of the additional stability of RNA complexes is twofold: there is a slightly attenuated energetic penalty for ring closure in the formation of RNA bidentate chelation complexes and elevated electrostatic interactions between the RNA and cations. In sum, it can be seen why sodium and calcium cannot replicate the structures or energetics of RNA-magnesium complexes.

The Ribosome Challenge to the RNA World

An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3′ end. Here we use selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3′ end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.

Molecular paleontology: a biochemical model of the ancestral ribosome

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

Functional RNAs: combined assembly and packaging in VLPs

Abstract We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qβ. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qβ virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qβ hairpin on the 5΄ end confers affinity for the Qβ coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP–RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP–RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qβ VLP packaging is demonstrated to be target-specific and efficient.

Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event

Abstract Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions.

Yeast rRNA Expansion Segments: Folding and Function

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome. Here, we characterize the binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7), which is the largest and most variable ES of the eukaryotic large ribosomal subunit and is located at the surface of the ribosome. In vitro RNA-protein pull-down experiments using ES7 as a bait indicate that ES7 is a binding hub for a variety of non-ribosomal proteins essential to ribosomal function in eukaryotes. ES7-associated proteins observed here cluster into four groups based on biological process, (i) response to abiotic stimulus (e.g., response to external changes in temperature, pH, oxygen level, etc.), (ii) ribosomal large subunit biogenesis, (iii) protein transport and localization, and (iv) transcription elongation. Seven synthetases, Ala-, Arg-, Asp-, Asn-, Leu-, Lys- and TyrRS, appear to associate with ES7. Affinities of AspRS, TyrRS and LysRS for ES7 were confirmed by in vitro binding assays. The results suggest that ES7 in S. cerevisiae could play a role analogous to the multi-synthetase complex present in higher order organisms and could be important for the appropriate function of the ribosome. Thermal denaturation studies and footprinting experiments confirm that isolated ES7 is stable and maintains a near-native secondary and tertiary structure.

Multiple prebiotic metals mediate translation

ABSTRACT Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O 2 ,high Fe 2+ , high Mn 2+ ) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg 2+ vs. Fe 2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe 2+ or Mn 2+ could mediate protein production, and quantified ribosomal metal content. We found that: (i) Fe 2+ and Mg 2+ had strikingly similar effects on rRNA folding; (ii) Fe 2+ and Mn 2+ can replace Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe 2+ and Mn 2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe 2+ and Mn 2+ were abundant, these findings suggest that Fe 2+ and Mn 2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O 2 , high Fe 2+ , high Mn 2+ ). Our results expand the roles of Fe 2+ and Mn 2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe2+ in life, despite its insolubility in the presence of oxygen, appears to stem from conditions of the ancient Earth. Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe2+ can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg2+ vs. Fe2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ could mediate protein production, and quantified ribosomal iron content. We found that: (i) Fe2+ and Mg2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe2+ was abundant, these findings suggest that Fe2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organisms where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and Fe2+-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions, (high-Fe2+, low-O2). Our results expand the role of Fe2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe 2+ in life, despite its toxicity and insolubility, appears to stem from conditions of the ancient Earth. Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life9s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe 2+ can revert the ribosome to a functional ancestral state. We find that Fe 2+ associates extensively with the ribosome and replaces Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein. Given that the translation system originated and matured when Fe 2+ was abundant, these findings suggest that Fe 2+ played a role in early ribosomal evolution.

Ferrous iron folds rRNA and mediates translation

ABSTRACT Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O 2 ,high Fe 2+ , high Mn 2+ ) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg 2+ vs. Fe 2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe 2+ or Mn 2+ could mediate protein production, and quantified ribosomal metal content. We found that: (i) Fe 2+ and Mg 2+ had strikingly similar effects on rRNA folding; (ii) Fe 2+ and Mn 2+ can replace Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe 2+ and Mn 2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe 2+ and Mn 2+ were abundant, these findings suggest that Fe 2+ and Mn 2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O 2 , high Fe 2+ , high Mn 2+ ). Our results expand the roles of Fe 2+ and Mn 2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe2+ in life, despite its insolubility in the presence of oxygen, appears to stem from conditions of the ancient Earth. Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe2+ can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg2+ vs. Fe2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ could mediate protein production, and quantified ribosomal iron content. We found that: (i) Fe2+ and Mg2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe2+ was abundant, these findings suggest that Fe2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organisms where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and Fe2+-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions, (high-Fe2+, low-O2). Our results expand the role of Fe2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe 2+ in life, despite its toxicity and insolubility, appears to stem from conditions of the ancient Earth. Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life9s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe 2+ can revert the ribosome to a functional ancestral state. We find that Fe 2+ associates extensively with the ribosome and replaces Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein. Given that the translation system originated and matured when Fe 2+ was abundant, these findings suggest that Fe 2+ played a role in early ribosomal evolution.