Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Broken symmetry between RNA enantiomers in a crystal lattice.

Explaining the origin of the homochirality of biological molecules requires a mechanism of disrupting the natural equilibrium between enantiomers and amplifying the initial imbalance to significant levels. Authors of existing models have sought an explanation in the parity-breaking weak nuclear force, in some selectively acting external factor, or in random fluctuations that subsequently became amplified by an autocatalytic process. We have obtained crystals in which l- and d-enantiomers of short RNA duplexes assemble in an asymmetric manner. These enantiomers make different lattice contacts and have different exposures to water and metal ions present in the crystal. Apparently, asymmetry between enantiomers can arise upon their mutual interactions and then propagate via crystallization. Asymmetric racemic compounds are worth considering as possible factors in symmetry breaking and enantioenrichment that took place in the early biosphere.

Chaperoning 5S RNA assembly.

In eukaryotes, three of the four ribosomal RNAs (rRNAs)­the 5.8S, 18S, and 25S/28S rRNAs­are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.

Molecular Dynamics Simulations of Reduced and Oxidized TFIIIA Zinc Fingers Free and Interacting with 5S RNA.

Interactions of zinc finger (ZF) proteins with nucleic acids and proteins play an important role in DNA transcription and repair, biochemical recognition, and protein regulation. The release of Zn2+ through oxidation of cysteine thiolates is associated with disruption of gene expression and DNA repair, preventing tumor growth. Multi-microsecond molecular dynamics (MD) simulations were carried out to examine the effect of Cys oxidation on the ZF456 fragment of transcription factor III A (TFIIIA) and its complex with 5S RNA. In the absence of 5S RNA, the reduced ZF456 peptide undergoes conformational changes in the secondary structure due to the reorientation of the intact ZF domains. Upon oxidation, the individual ZF domains unfold to various degrees, yielding a globular ZF456 peptide with ZF4 and ZF6, responsible for base-specific hydrogen bonds with 5S RNA, losing their ßßα-folds. ZF5, on the other hand, participates in nonspecific interactions through its α-helix that conditionally unravels early in the simulation. In the presence of RNA, oxidation of the ZF456 peptide disrupts the key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. However, interactions with ZF4 are dependent on the protonation state of His119.

Mirror-Image 5S Ribonucleoprotein Complexes.

After realizing mirror-image genetic replication, transcription, and reverse transcription, the biggest challenge in establishing a mirror-image version of the central dogma is to build a mirror-image ribosome-based translation machine. Here, we chemically synthesized the natural and mirror-image versions of three ribosomal proteins (L5, L18, and L25) in the large subunit of the Escherichia coli ribosome with post-translational modifications. We show that the synthetic mirror-image proteins can fold in vitro despite limited efficiency and assemble with enzymatically transcribed mirror-image 5S ribosomal RNA into ribonucleoprotein complexes. In addition, the RNA-protein interactions are chiral-specific in that the mirror-image ribosomal proteins do not bind with natural 5S ribosomal RNA and vice versa. The synthesis and assembly of mirror-image 5S ribonucleoprotein complexes are important steps towards building a functional mirror-image ribosome.

Advanced multi-loop algorithms for RNA secondary structure prediction reveal that the simplest model is best.

Algorithmic prediction of RNA secondary structure has been an area of active inquiry since the 1970s. Despite many innovations since then, our best techniques are not yet perfect. The workhorses of the RNA secondary structure prediction engine are recursions first described by Zuker and Stiegler in 1981. These have well understood caveats; a notable flaw is the ad-hoc treatment of multi-loops, also called helical-junctions, that persists today. While several advanced models for multi-loops have been proposed, it seems to have been assumed that incorporating them into the recursions would lead to intractability, and so no algorithms for these models exist. Some of these models include the classical model based on Jacobson-Stockmayer polymer theory, and another by Aalberts and Nadagopal that incorporates two-length-scale polymer physics. We have realized practical, tractable algorithms for each of these models. However, after implementing these algorithms, we found that no advanced model was better than the original, ad-hoc model used for multi-loops. While this is unexpected, it supports the praxis of the current model.

5SRNAdb: an information resource for 5S ribosomal RNAs.

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces.

Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.

Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure-function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA-RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA-RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.

Structural and functional analysis of the Rpf2-Rrs1 complex in ribosome biogenesis.

Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The long α-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a ß-barrel shape with anti-parallel ß-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120 bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.

Molecular organization of the 5S rDNA gene type II in elasmobranchs.

The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.

Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae.

Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.

Organization and variation analysis of 5S rDNA in gynogenetic offspring of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂).

BACKGROUND: The offspring with 100 chromosomes (abbreviated as GRCC) have been obtained in the first generation of Carassius auratus red var. (abbreviated as RCC, 2n = 100) (♀) × Megalobrama amblycephala (abbreviated as BSB, 2n = 48) (♂), in which the females and unexpected males both are found. Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested, but lack genetic evidence. RESULT: Fluorescence in situ hybridization with species-specific centromere probes directly proves that GRCC possess two sets of RCC-derived chromosomes. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (abbreviated as NTS) reveals that three types of 5S rDNA class (class I; class II and class III) in GRCC are completely inherited from their female parent (RCC), and show obvious base variations and insertions-deletions. Fluorescence in situ hybridization with the entire 5S rDNA probe reveals obvious chromosomal loci (class I and class II) variation in GRCC. CONCLUSIONS: This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our result is also reveals that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.

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).

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species.

Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Statistical evaluation of improvement in RNA secondary structure prediction.

With discovery of diverse roles for RNA, its centrality in cellular functions has become increasingly apparent. A number of algorithms have been developed to predict RNA secondary structure. Their performance has been benchmarked by comparing structure predictions to reference secondary structures. Generally, algorithms are compared against each other and one is selected as best without statistical testing to determine whether the improvement is significant. In this work, it is demonstrated that the prediction accuracies of methods correlate with each other over sets of sequences. One possible reason for this correlation is that many algorithms use the same underlying principles. A set of benchmarks published previously for programs that predict a structure common to three or more sequences is statistically analyzed as an example to show that it can be rigorously evaluated using paired two-sample t-tests. Finally, a pipeline of statistical analyses is proposed to guide the choice of data set size and performance assessment for benchmarks of structure prediction. The pipeline is applied using 5S rRNA sequences as an example.