Characterization of agapornis fischeri interferon gamma and its activity against beak and feather disease virus.

This study sought to clone and sequence the interferon-γ (IFN-γ) gene of the Fischer's lovebird parrot (Agapornis fischeri). Raw264.7 cells treated with the expressed IFN-γ protein exhibited an upregulation in inducible nitric oxide synthase protein expression and nitric oxide (NO) production coupled with increases in phagocytosis and pinocytosis, as well as an induction of interferon-stimulated genes through the activation of the NF-κB factor, all of which are indicators of the innate immune responses of the activated macrophages. Similar to the IFN-γ protein of other species, the NO production activity of the parrot IFN-γ protein decreased by 80% after exposure at 60 °C for 4 min. Additionally, only half of the NO production activity of the parrot IFN-γ protein remained upon exposure to HCl for 30 min. These findings suggested that the parrot IFN-γ protein was heat-labile and sensitive to acidic conditions. Therefore, all of these effects contributed to the blockage of the uptake of BFDV virus-like particles (VLPs) by cells, the nuclear entry of the Cap protein of BFDV VLPs, and the clearance of the virus from BFDV-infected parrots by the IFN-γ protein of Agapornis fischeri. This study is the first to describe the cloning of the IFN-γ gene of Agapornis fischeri and characterize the anti-beak and feather disease virus activity of the IFN-γ protein of Agapornis fischeri.

Characterization of the nuclear localization sequence of beak and feather disease virus capsid proteins and their assembly into virus-like particles.

Beak and feather disease virus (BFDV) is a single-stranded circular DNA icosahedral virus that belongs to the Circoviridae family. This virus is the causative pathogen of beak and feather disease, which leads to feather loss, malformed claws, and immunosuppression of psittacine birds. Our study produced BFDV virus-like particles (VLPs) including capsid proteins, mutant Cap proteins (Cap ΔNLS54, Cap ΔNLS62, Cap C228S, and Cap ΔNES) and chimeric Cap proteins carrying the epitope (amino acid residues 64-70) of the replication-associated protein (R-Cap, Cap-R, R-Cap ΔNLS54, and Cap ΔNLS54-R). All of the aforementioned VLPs were observed via transmission electron microscopy and verified through immunogold labeling. The nuclear localization sequence (NLS) of the Cap protein was identified between amino acid residues 55-62. Nuclear export of the Cap protein depended on the nuclear export sequence (NES). All VLPs except Cap ΔNLS62 and Cap ΔNES entered the cells 2 h post-infection (hpi) and were shuttled into the nucleus at 8 hpi. Wheat germ agglutinin (WGA) blocked the nuclear entry of Cap proteins at 8 hpi and the nuclear export of Cap proteins at 16 hpi was inhibited by leptomycin B. The nuclear entry of Cap protein was inhibited by importin α and importin ß inhibitors, as well as NLS peptides. Moreover, the interactions of Cap proteins and Cap VLPs with both importin α and importin ß were characterized via the GST pull-down and immunofluorescence assays. These interactions were blocked by the presence of importin α and importin ß inhibitors, as well as NLS peptides. Therefore, our study is the first to describe the precise position of the NLS of the BFDV Cap protein and the interaction of Cap protein with importin α and importin ß in vitro.

The study of isotopic enrichment of water in human plasma and erythrocyte.

Life does not sustain without water. For water, there is a natural abundance of stable isotope hydrogen and oxygen. Water molecules get across cell membranes through a plasma membrane protein, named aquaporin. Moreover, the kidney is the main organ to maintain water homeostasis. Here, we study the stable isotopic ratios of hydrogen and oxygen in human blood plasma and erythrocyte corresponding to kidney functions. We extract waters from human plasma and erythrocyte, collected from 110 participants, including 51 clinically stable outpatients with end-stage renal disease (ESRD) and 59 subjects with normal renal function (NRF). We observed that (i) both extracellular (blood plasma) and intracellular (erythrocyte) biology waters are isotopic differences between the ESRD and NRF participants, (ii) the natural abundance of isotopic waters of ESRD is hypo-isotopic, and (iii) the isotopic enrichment of water between erythrocyte and blood plasma are distinct. In addition, we introduce an empirical formula using entropy transformation to describe isotopic water enrichment for biology. Accordingly, the natural abundance of stable isotope water of blood plasma and erythrocyte may be possibly put in practice a new sign for assessments of kidney dysfunctions.

Characterization of the endonuclease activity of the replication-associated protein of beak and feather disease virus.

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.

Prebiotic Iron Originates the Peptidyl Transfer Origin.

The ribosome is responsible for protein synthesis in all living organisms. It is best known to exist around 3.5-3.7 Ga whereat life on Earth inhabited anoxic environment with abundant soluble irons. The RNAs and proteins are the two biopolymers that constitute the ribosome. However, both proteins and RNAs require metal cations to fold and to function. There are four Mg-microcluster (Mg2+-µc) structures conserved in core of large subunit, and the 23S ribosomal RNA (rRNA) was shown to catalyze electron transfer in an anoxic environment in the presence of Fe2+. The Mg2+-µc features two idiosyncratic Mg2+ ions that are chelated and bridged by a common phosphate group and along with that, the adjacent residues of RNA backbone together forming ten-membered chelation ring(s). Here, we utilized four rRNA fragments of the large subunit 23S rRNA of Haloarcula marismortui, that includes the residues that form the four Mg2+-µc's. These four rRNA fragments are shown competent to assemble with Mg2+. Our results show that when these rRNA fragments fold or assembly in the presence of Fe2+ under anoxic conditions, each Fe2+-microcluster can catalyze electron transfer. We propose that Fe2+-microclusters of the ribosome, which use Fe2+ as a cofactor to regulate electron transfer, are pivotal and primordial and may be an origin in evolution of the ribosome.

RNA: packaged and protected by VLPs.

Virus Like Particles (VLPs) are devices for RNA packaging, protection and delivery, with utility in fundamental research, drug discovery, and disease treatment. Using E. coli for combined expression and packaging of non-viral RNAs into Qß VLPs, we investigated the extent of chemical protection conferred by packaging of RNA in VLPs. We also probed relationships between packaging efficiency and RNA size, sequence and intrinsic compaction. We observe that VLP packaging protects RNA against assault by small diffusible damaging agents such as hydroxyl radicals and divalent cations. By contrast, the extent of unmediated cleavage, in the absence of reactive species, is the same for RNA that is free or packaged within VLPs, and is very slow. In vivo packaging of RNA within VLPs appears to be more efficient for intrinsically compact RNAs, such as rRNA, and less efficient for unstructured, elongated RNA such as mRNA. Packaging efficiency is reduced by addition of the ribosome binding site to a target RNA. The Qß hairpin is necessary but not sufficient for efficient packaging.

Functional RNAs: combined assembly and packaging in VLPs.

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qß. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qß 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 Qß hairpin on the 5΄ end confers affinity for the Qß 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 Qß VLP packaging is demonstrated to be target-specific and efficient.

RiboVision suite for visualization and analysis of ribosomes.

RiboVision is a visualization and analysis tool for the simultaneous display of multiple layers of diverse information on primary (1D), secondary (2D), and three-dimensional (3D) structures of ribosomes. The ribosome is a macromolecular complex containing ribosomal RNA and ribosomal proteins and is a key component of life responsible for the synthesis of proteins in all living organisms. RiboVision is intended for rapid retrieval, analysis, filtering, and display of a variety of ribosomal data. Preloaded information includes 1D, 2D, and 3D structures augmented by base-pairing, base-stacking, and other molecular interactions. RiboVision is preloaded with rRNA secondary structures, rRNA domains and helical structures, phylogeny, crystallographic thermal factors, etc. RiboVision contains structures of ribosomal proteins and a database of their molecular interactions with rRNA. RiboVision contains preloaded structures and data for two bacterial ribosomes (Thermus thermophilus and Escherichia coli), one archaeal ribosome (Haloarcula marismortui), and three eukaryotic ribosomes (Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens). RiboVision revealed several major discrepancies between the 2D and 3D structures of the rRNAs of the small and large subunits (SSU and LSU). Revised structures mapped with a variety of data are available in RiboVision as well as in a public gallery (). RiboVision is designed to allow users to distill complex data quickly and to easily generate publication-quality images of data mapped onto secondary structures. Users can readily import and analyze their own data in the context of other work. This package allows users to import and map data from CSV files directly onto 1D, 2D, and 3D levels of structure. RiboVision has features in rough analogy with web-based map services capable of seamlessly switching the type of data displayed and the resolution or magnification of the display. RiboVision is available at .

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Secondary structures of rRNAs from all three domains of life.

Accurate secondary structures are important for understanding ribosomes, which are extremely large and highly complex. Using 3D structures of ribosomes as input, we have revised and corrected traditional secondary (2°) structures of rRNAs. We identify helices by specific geometric and molecular interaction criteria, not by co-variation. The structural approach allows us to incorporate non-canonical base pairs on parity with Watson-Crick base pairs. The resulting rRNA 2° structures are up-to-date and consistent with three-dimensional structures, and are information-rich. These 2° structures are relatively simple to understand and are amenable to reproduction and modification by end-users. The 2° structures made available here broadly sample the phylogenetic tree and are mapped with a variety of data related to molecular interactions and geometry, phylogeny and evolution. We have generated 2° structures for both large subunit (LSU) 23S/28S and small subunit (SSU) 16S/18S rRNAs of Escherichia coli, Thermus thermophilus, Haloarcula marismortui (LSU rRNA only), Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. We provide high-resolution editable versions of the 2° structures in several file formats. For the SSU rRNA, the 2° structures use an intuitive representation of the central pseudoknot where base triples are presented as pairs of base pairs. Both LSU and SSU secondary maps are available (http://apollo.chemistry.gatech.edu/RibosomeGallery). Mapping of data onto 2° structures was performed on the RiboVision server (http://apollo.chemistry.gatech.edu/RiboVision).

Secondary structure and domain architecture of the 23S and 5S rRNAs.

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).

RNA with iron(II) as a cofactor catalyses electron transfer.

Mg(2+) is essential for RNA folding and catalysis. However, for the first 1.5 billion years of life on Earth RNA inhabited an anoxic Earth with abundant and benign Fe(2+). We hypothesize that Fe(2+) was an RNA cofactor when iron was abundant, and was substantially replaced by Mg(2+) during a period known as the 'great oxidation', brought on by photosynthesis. Here, we demonstrate that reversing this putative metal substitution in an anoxic environment, by removing Mg(2+) and replacing it with Fe(2+), expands the catalytic repertoire of RNA. Fe(2+) can confer on some RNAs a previously uncharacterized ability to catalyse single-electron transfer. We propose that RNA function, in analogy with protein function, can be understood fully only in the context of association with a range of possible metals. The catalysis of electron transfer, requisite for metabolic activity, may have been attenuated in RNA by photosynthesis and the rise of O2.

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.

RNA-magnesium-protein interactions in large ribosomal subunit.

Some of the magnesium ions in the ribosome are coordinated by multiple rRNA phosphate groups. These magnesium ions link distal sequences of rRNA, primarily by incorporating phosphate groups into the first coordination shell. Less frequently, magnesium interacts with ribosomal proteins. Ribosomal protein L2 appears to be unique by forming specific magnesium-mediated interactions with rRNA. Using optimized models derived from X-ray structures, we subjected rRNA/magnesium/water/rProtein L2 assemblies to quantum mechanical analysis using the density functional theory and natural energy decomposition analysis. The combined results provide estimates of energies of formation of these assemblies, and allow us to decompose the energies of interaction. The results indicated that RNA immobilizes magnesium by multidentate chelation with phosphate, and that the magnesium ions in turn localize and polarize water molecules, increasing energies and specificities of interaction of these water molecules with L2 protein. Thus, magnesium plays subtle, yet important, roles in ribosomal assembly beyond neutralization of electrostatic repulsion and direct coordination of RNA functional groups.

RNA folding and catalysis mediated by iron (II).

Mg²âº 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 Fe²âº in the absence of free oxygen as a replacement for Mg²âº in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²âº in RNA folding and function can indeed be served by Fe²âº. The results of quantum mechanical calculations show that the geometry of coordination of Fe²âº by RNA phosphates is similar to that of Mg²âº. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²âº or Mg²âº. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²âº, and the hammerhead ribozyme are enhanced in the presence of Fe²âº compared to Mg²âº. All chemical footprinting and ribozyme assays in the presence of Fe²âº 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 Fe²âº. The combined biochemical and paleogeological data are consistent with a role for Fe²âº in an RNA World. RNA and Fe²âº could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²âº alone.

Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state.

The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.

B-DNA structure is intrinsically polymorphic: even at the level of base pair positions.

Increasingly exact measurement of single crystal X-ray diffraction data offers detailed characterization of DNA conformation, hydration and electrostatics. However, instead of providing a more clear and unambiguous image of DNA, highly accurate diffraction data reveal polymorphism of the DNA atomic positions and conformation and hydration. Here we describe an accurate X-ray structure of B-DNA, painstakingly fit to a multistate model that contains multiple competing positions of most of the backbone and of entire base pairs. Two of ten base-pairs of CCAGGCCTGG are in multiple states distinguished primarily by differences in slide. Similarly, all the surrounding ions are seen to fractionally occupy discrete competing and overlapping sites. And finally, the vast majority of water molecules show strong evidence of multiple competing sites. Conventional resolution appears to give a false sense of homogeneity in conformation and interactions of DNA. In addition, conventional resolution yields an average structure that is not accurate, in that it is different from any of the multiple discrete structures observed at high resolution. Because base pair positional heterogeneity has not always been incorporated into model-building, even some high and ultrahigh-resolution structures of DNA do not indicate the full extent of conformational polymorphism.

RNA tetraloop folding reveals tension between backbone restraints and molecular interactions.

In RNA, A-form helices are commonly terminated by tetraloops or 3' dangling ends. Aside from helices themselves, these helix-breaking motifs appear to be among the most frequent and repetitive structural elements of large folded RNAs. We show here that within a frequent type of tetraloop, cGNRAg (G is guanine, N is any base, R is purine, A is adenine), a tension exists between the backbone torsional energy of the loop and the energy contributed by molecular interactions (stacking and pairing). A model in which favorable bond rotamers are opposed by favorable stacking and pairing interactions is consistent with our observation that release of torsional restraints upon conversion of one or more loop riboses to more flexible trimethylene phosphate(s) contributes favorably to the enthalpy of folding. This effect presumably results from improved stacking and hydrogen-bonding interactions upon release of torsional restraints. The most obvious possibility for improving molecular interactions is a repositioning of A, which is proximal to the unfavorable torsion angles in native cGNRAg tetraloops, and which is unstacked on the 3' side and unpaired (it forms a single hydrogen bond with the opposing G). This tension between favorable bond rotamers and favorable molecular interactions may be representative of a general evolutionary strategy to prevent achievement of deep and irreversible thermodynamic wells in folded RNAs. Finally, we observe a simple stacking substructure with conserved geometry and sequence that forms a scaffold for both tetraloops and 3' dangling ends. It seems that simple substructures can build RNA motifs, which combine to establish the fundamental architecture of RNA.

Mechanism of RNA double helix-propagation at atomic resolution.

The conversion of a nucleic acid from single strands to double strands is thought to involve slow nucleation followed by fast double-strand propagation. Here, for RNA double-strand propagation, we propose an atomic resolution reaction mechanism. This mechanism, called the stack-ratchet, is based on data-mining of three-dimensional structures and on available thermodynamic information. The stack-ratchet mechanism extends and adds detail to the classic zipper model proposed by Porschke (Porschke, D. Biophysical Chemistry 1974, 2, pp. 97-101). Porschke's zipper model describes the addition of a base pair to a nucleated helix in terms of a single type of elementary reaction; a concerted process in which the two bases, one from each strand, participate in the transition state. In the stack-ratchet mechanism proposed here a net base-pairing step consists of two elementary reactions. Motions of only one strand are required to achieve a given transition state. One elementary reaction preorganizes and stacks the 3' single-strand, driven by base--base stacking interactions. A second elementary reaction stacks the 5' strand and pairs it with the preorganized 3' strand. In the stack-ratchet mechanism, a variable length 3' stack leads the single-strand/double-strand junction. The stack-ratchet mechanism is not a two-state process. A base can be (i) unstacked and unpaired, (ii) stacked and paired, or (ii) stacked and unpaired (only on the 3' strand). The data suggests that helices of DNA and of RNA do not propagate by similar mechanisms.

Peeling the onion: ribosomes are ancient molecular fossils.

We describe a method to establish chronologies of ancient ribosomal evolution. The method uses structure-based and sequence-based comparison of the large subunits (LSUs) of Haloarcula marismortui and Thermus thermophilus. These are the highest resolution ribosome structures available and represent disparate regions of the evolutionary tree. We have sectioned the superimposed LSUs into concentric shells, like an onion, using the site of peptidyl transfer as the origin (the PT-origin). This spherical approximation combined with a shell-by-shell comparison captures significant information along the evolutionary time line revealing, for example, that sequence and conformational similarity of the 23S rRNAs are greatest near the PT-origin and diverge smoothly with distance from it. The results suggest that the conformation and interactions of both RNA and protein can be described as changing, in an observable manner, over evolutionary time. The tendency of macromolecules to assume regular secondary structural elements such as A-form helices with Watson-Crick base pairs (RNA) and alpha-helices and beta-sheets (protein) is low at early time points but increases as time progresses. The conformations of ribosomal protein components near the PT-origin suggest that they may be molecular fossils of the peptide ancestors of ribosomal proteins. Their abbreviated length may have proscribed formation of secondary structure, which is indeed nearly absent from the region of the LSU nearest the PT-origin. Formation and evolution of the early PT center may have involved Mg(2+)-mediated assembly of at least partially single-stranded RNA oligomers or polymers. As one moves from center to periphery, proteins appear to replace magnesium ions. The LSU is known to have undergone large-scale conformation changes upon assembly. The T. thermophilus LSU analyzed here is part of a fully assembled ribosome, whereas the H. marismortui LSU analyzed here is dissociated from other ribosomal components. Large-scale conformational differences in the 23S rRNAs are evident from superimposition and prevent structural alignment of some portions of the rRNAs, including the L1 stalk.