RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.histidine relaxase | antibiotic resistance | horizontal gene transfer | X-ray structure | Staphylococcus aureus A cquisition of exogenous genetic material by bacteria is achieved via conjugative DNA transfer of mobile genetic elements, such as plasmids and especially integrative and conjugative elements and integrative and mobilizable elements (1). Such processes of horizontal gene transfer (HGT) are considered a strong driving force in bacterial evolution and in the ability of bacteria to colonize different niches (2). In addition to permitting the rapid evolution of the bacterial pangenome, HGT is involved in the acquisition of genetic traits that may confer selective advantages to the recipient bacteria, including antibiotic resistance (3). This is particularly important when resistance genes encoded by mobile elements are spread explosively among bacteria in hospitals, posing a serious threat to public health systems (www.cdc.gov/drugresistance/threat-report-2013; www. who.int/drugresistance/documents/surveillancereport/en/). Thus, the so-called mobilome (4) participates in the spread of antibiotic resistance, which is expected to cause 10 million casualties annually by 2050, and the consequent huge economic burden (amr-review.org/sites/default/files/Report-52.15.pdf). This has generated a unanimous call for new approaches to deal with infectious diseases caused by pathogenic bacteria (5).A main performer in HGT is the protein relaxase, a topoisomeraselike enzyme that cleaves supercoiled plasmid DNA in a strandand sequence-specific manner and ligates it after cell-to-cell transfer. Relaxases start DNA transfer by conjugation on recognition of their target DNA, the origin of transfer (oriT), on which they mediate generation of a hairpin-loop structure that leaves the dinucleotide to be cleaved (the nic site) in a singlestranded (ss) DNA configuration (Fig. 1B) (6, 7). On the oriT, the relaxase assembles with other proteins p...

RNArchitecture is a database that provides a comprehensive description of relationships between known families of structured non-coding RNAs, with a focus on structural similarities. The classification is hierarchical and similar to the system used in the SCOP and CATH databases of protein structures. Its central level is Family, which builds on the Rfam catalog and gathers closely related RNAs. Consensus structures of Families are described with a reduced secondary structure representation. Evolutionarily related Families are grouped into Superfamilies. Similar structures are further grouped into Architectures. The highest level, Class, organizes families into very broad structural categories, such as simple or complex structured RNAs. Some groups at different levels of the hierarchy are currently labeled as ‘unclassified’. The classification is expected to evolve as new data become available. For each Family with an experimentally determined three-diemsional (3D) structure(s), a representative one is provided. RNArchitecture also presents theoretical models of RNA 3D structure and is open for submission of structural models by users. Compared to other databases, RNArchitecture is unique in its focus on structure-based RNA classification, and in providing a platform for storing RNA 3D structure predictions. RNArchitecture can be accessed at http://iimcb.genesilico.pl/RNArchitecture/.