Expression, Purification, and Analysis of Unknown Translation Factors from Escherichia coli: A Synthesis Approach

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation

Structure of human serum cholinesterase

Human cholinesterase has recently been sequenced and cloned. It is a glycoprotein of 4 identical subunits, each subunit containing 9 carbohydrate chains and 3.5 disulfide bonds. Protein folding is likely to be very similar in human cholinesterase and Torpedo acetylcholinesterase. The cholinesterases have no significant sequence homology with the serine proteases and seem to belong to a separate serine esterase family.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50189/1/950090406_ftp.pd

Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg2+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg2+. Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons

Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures

An analysis of simple computational strategies to facilitate the design of functional molecular information processors

BACKGROUND: Biological macromolecules (DNA, RNA and proteins) are capable of processing physical or chemical inputs to generate outputs that parallel conventional Boolean logical operators. However, the design of functional modules that will enable these macromolecules to operate as synthetic molecular computing devices is challenging. RESULTS: Using three simple heuristics, we designed RNA sensors that can mimic the function of a seven-segment display (SSD). Ten independent and orthogonal sensors representing the numerals 0 to 9 are designed and constructed. Each sensor has its own unique oligonucleotide binding site region that is activated uniquely by a specific input. Each operator was subjected to a stringent in silico filtering. Random sensors were selected and functionally validated via ribozyme self cleavage assays that were visualized via electrophoresis. CONCLUSIONS: By utilising simple permutation and randomisation in the sequence design phase, we have developed functional RNA sensors thus demonstrating that even the simplest of computational methods can greatly aid the design phase for constructing functional molecular devices. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-016-1297-x) contains supplementary material, which is available to authorized users

Non Coding RNAs and Viruses in the Framework of the Phylogeny of the Genes, Epigenesis and Heredity

The origin of genes is one of the most enigmatic events in the origin of life. It has been suggested that noncoding (nc) RNA was probably a precursor in the formation of the first polypeptide, and also at the origin of the first manifestation of life and genes. ncRNAs are also becoming central for understanding gene expression and silencing. Indeed, before the discovery of ncRNAs, proteins were viewed as the major molecules in the regulation of gene expression and gene silencing; however, recent findings suggest that ncRNA also plays an important role in gene expression. Reverse transcription of RNA viruses and their integration into the genome of eukaryotes and also their relationship with the ncRNA suggest that their origin is basal in genome evolution, and also probably constitute the first mechanism of gene regulation. I am to review the different roles of ncRNAs in the framework of gene evolution, as well as the importance of ncRNAs and viruses in the epigenesis and in the non-Mendelian model of heredity and evolution

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

[EN] Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplastreplicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.This work was supported by the European Research Council (erc.europa.eu; ERC-2011-StG-281191-VIRMUT to RS), the Spanish Ministerio de Economia y Competitividad (www.mineco.gob.es; BFU2013-41329 grant to RS, BFU2014-56812-P grant to RF, and a predoctoral fellowship to ALC), and the Spanish Junta de Comunidades de Castilla-La Mancha (www.castillalamancha.es;postdoctoral fellowship to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Carrasco, MA.; Ballesteros Martínez, C.; Sentandreu, V.; Delgado Villar, SG.; Gago Zachert, SP.; Flores Pedauye, R.; Sanjuan Verdeguer, R. (2017). Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing. PLoS Pathogens. 13(9):1-17. https://doi.org/10.1371/journal.ppat.1006547S117139Ganai, R. A., & Johansson, E. (2016). DNA Replication—A Matter of Fidelity. Molecular Cell, 62(5), 745-755. doi:10.1016/j.molcel.2016.05.003Lynch, M. (2010). Evolution of the mutation rate. Trends in Genetics, 26(8), 345-352. doi:10.1016/j.tig.2010.05.003Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and Molecular Life Sciences, 73(23), 4433-4448. doi:10.1007/s00018-016-2299-6Gago, S., Elena, S. F., Flores, R., & Sanjuan, R. (2009). Extremely High Mutation Rate of a Hammerhead Viroid. Science, 323(5919), 1308-1308. doi:10.1126/science.1169202Flores, R., Gago-Zachert, S., Serra, P., Sanjuán, R., & Elena, S. F. (2014). Viroids: Survivors from the RNA World? Annual Review of Microbiology, 68(1), 395-414. doi:10.1146/annurev-micro-091313-103416Flores, R., Minoia, S., Carbonell, A., Gisel, A., Delgado, S., López-Carrasco, A., … Di Serio, F. (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research, 209, 136-145. doi:10.1016/j.virusres.2015.02.027Steger, G., & Perreault, J.-P. (2016). Structure and Associated Biological Functions of Viroids. Advances in Virus Research, 141-172. doi:10.1016/bs.aivir.2015.11.002Diener, T. O. (1989). Circular RNAs: relics of precellular evolution? Proceedings of the National Academy of Sciences, 86(23), 9370-9374. doi:10.1073/pnas.86.23.9370Ambrós, S., Hernández, C., & Flores, R. (1999). Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host The data reported in this paper are in the EMBL nucleotide sequence database and assigned the accession nos AJ241818–AJ241850. Journal of General Virology, 80(8), 2239-2252. doi:10.1099/0022-1317-80-8-2239Navarro, J.-A., Vera, A., & Flores, R. (2000). A Chloroplastic RNA Polymerase Resistant to Tagetitoxin Is Involved in Replication of Avocado Sunblotch Viroid. Virology, 268(1), 218-225. doi:10.1006/viro.1999.0161Rodio, M.-E., Delgado, S., De Stradis, A., Gómez, M.-D., Flores, R., & Di Serio, F. (2007). A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development. The Plant Cell, 19(11), 3610-3626. doi:10.1105/tpc.106.049775Carbonell, A., De la Peña, M., Flores, R., & Gago, S. (2006). Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads. Nucleic Acids Research, 34(19), 5613-5622. doi:10.1093/nar/gkl717Hutchins, C. J., Rathjen, P. D., Forster, A. C., & Symons, R. H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Research, 14(9), 3627-3640. doi:10.1093/nar/14.9.3627PRODY, G. A., BAKOS, J. T., BUZAYAN, J. M., SCHNEIDER, I. R., & BRUENING, G. (1986). Autolytic Processing of Dimeric Plant Virus Satellite RNA. Science, 231(4745), 1577-1580. doi:10.1126/science.231.4745.1577Nohales, M.-A., Molina-Serrano, D., Flores, R., & Daros, J.-A. (2012). Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae. Journal of Virology, 86(15), 8269-8276. doi:10.1128/jvi.00629-12Branch, A. D., Benenfeld, B. J., & Robertson, H. D. (1988). Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 85(23), 9128-9132. doi:10.1073/pnas.85.23.9128Daros, J.-A., & Flores, R. (2004). Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceedings of the National Academy of Sciences, 101(17), 6792-6797. doi:10.1073/pnas.0401090101Feldstein, P. A., Hu, Y., & Owens, R. A. (1998). Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 95(11), 6560-6565. doi:10.1073/pnas.95.11.6560Gas, M.-E., Hernández, C., Flores, R., & Daròs, J.-A. (2007). Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations. PLoS Pathogens, 3(11), e182. doi:10.1371/journal.ppat.0030182Nohales, M.-A., Flores, R., & Daros, J.-A. (2012). Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proceedings of the National Academy of Sciences, 109(34), 13805-13810. doi:10.1073/pnas.1206187109Brass, J. R. J., Owens, R. A., Matoušek, J., & Steger, G. (2017). Viroid quasispecies revealed by deep sequencing. RNA Biology, 14(3), 317-325. doi:10.1080/15476286.2016.1272745Bull, J. J., Sanjuán, R., & Wilke, C. O. (2007). Theory of Lethal Mutagenesis for Viruses. Journal of Virology, 81(6), 2930-2939. doi:10.1128/jvi.01624-06Cuevas, J. M., González-Candelas, F., Moya, A., & Sanjuán, R. (2009). Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo. Journal of Virology, 83(11), 5760-5764. doi:10.1128/jvi.00201-09Ribeiro, R. M., Li, H., Wang, S., Stoddard, M. B., Learn, G. H., Korber, B. T., … Perelson, A. S. (2012). Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate. PLoS Pathogens, 8(8), e1002881. doi:10.1371/journal.ppat.1002881Acevedo, A., Brodsky, L., & Andino, R. (2013). Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature, 505(7485), 686-690. doi:10.1038/nature12861Cuevas, J. M., Geller, R., Garijo, R., López-Aldeguer, J., & Sanjuán, R. (2015). Extremely High Mutation Rate of HIV-1 In Vivo. PLOS Biology, 13(9), e1002251. doi:10.1371/journal.pbio.1002251Acevedo, A., & Andino, R. (2014). Library preparation for highly accurate population sequencing of RNA viruses. Nature Protocols, 9(7), 1760-1769. doi:10.1038/nprot.2014.118Kennedy, S. R., Schmitt, M. W., Fox, E. J., Kohrn, B. F., Salk, J. J., Ahn, E. H., … Loeb, L. A. (2014). Detecting ultralow-frequency mutations by Duplex Sequencing. Nature Protocols, 9(11), 2586-2606. doi:10.1038/nprot.2014.170Franklin, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proceedings of the National Academy of Sciences, 55(6), 1504-1511. doi:10.1073/pnas.55.6.1504López-Carrasco, A., Gago-Zachert, S., Mileti, G., Minoia, S., Flores, R., & Delgado, S. (2015). The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in theirin vivoRNA conformations. RNA Biology, 13(1), 83-97. doi:10.1080/15476286.2015.1119365Keese, P., & Symons, R. H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences, 82(14), 4582-4586. doi:10.1073/pnas.82.14.4582López-Carrasco, A., & Flores, R. (2016). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A «naked» rod-like conformation similar but not identical to that observed in vitro. RNA Biology, 14(8), 1046-1054. doi:10.1080/15476286.2016.1223005Flores, R., Hernandez, C., de la Peña, M., Vera, A., & Daros, J.-A. (2001). Hammerhead Ribozyme Structure and Function in Plant RNA Replication. Ribonucleases - Part A, 540-552. doi:10.1016/s0076-6879(01)41175-xMartick, M., & Scott, W. G. (2006). Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis. Cell, 126(2), 309-320. doi:10.1016/j.cell.2006.06.036Ruffner, D. E., Stormo, G. D., & Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry, 29(47), 10695-10702. doi:10.1021/bi00499a018Flores, R., Serra, P., Minoia, S., Di Serio, F., & Navarro, B. (2012). Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs. Frontiers in Microbiology, 3. doi:10.3389/fmicb.2012.00217Owens, R. A., Chen, W., Hu, Y., & Hsu, Y.-H. (1995). Suppression of Potato Spindle Tuber Viroid Replication and Symptom Expression by Mutations Which Stabilize the Pathogenicity Domain. Virology, 208(2), 554-564. doi:10.1006/viro.1995.1186Takeda, R., Petrov, A. I., Leontis, N. B., & Ding, B. (2011). A Three-Dimensional RNA Motif in Potato spindle tuber viroid Mediates Trafficking from Palisade Mesophyll to Spongy Mesophyll in Nicotiana benthamiana. The Plant Cell, 23(1), 258-272. doi:10.1105/tpc.110.081414Zhong, X., Leontis, N., Qian, S., Itaya, A., Qi, Y., Boris-Lawrie, K., & Ding, B. (2006). Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication. Journal of Virology, 80(17), 8566-8581. doi:10.1128/jvi.00837-06Zhong, X., Tao, X., Stombaugh, J., Leontis, N., & Ding, B. (2007). Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking. The EMBO Journal, 26(16), 3836-3846. doi:10.1038/sj.emboj.7601812Zhong, X., Archual, A. J., Amin, A. A., & Ding, B. (2008). A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking. The Plant Cell, 20(1), 35-47. doi:10.1105/tpc.107.056606Thomas, M. J., Platas, A. A., & Hawley, D. K. (1998). Transcriptional Fidelity and Proofreading by RNA Polymerase II. Cell, 93(4), 627-637. doi:10.1016/s0092-8674(00)81191-5Gout, J.-F., Thomas, W. K., Smith, Z., Okamoto, K., & Lynch, M. (2013). Large-scale detection of in vivo transcription errors. Proceedings of the National Academy of Sciences, 110(46), 18584-18589. doi:10.1073/pnas.1309843110Hedtke, B. (1997). Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis. Science, 277(5327), 809-811. doi:10.1126/science.277.5327.809Lerbs-Mache, S. (1993). The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes? Proceedings of the National Academy of Sciences, 90(12), 5509-5513. doi:10.1073/pnas.90.12.5509Oldenkott, B., Yamaguchi, K., Tsuji-Tsukinoki, S., Knie, N., & Knoop, V. (2014). Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyteSelaginella uncinata. RNA, 20(10), 1499-1506. doi:10.1261/rna.045575.114Codoñer, F. M., Darós, J.-A., Solé, R. V., & Elena, S. F. (2006). The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens. PLoS Pathogens, 2(12), e136. doi:10.1371/journal.ppat.0020136Eigen, M. (1971). Selforganization of matter and the evolution of biological macromolecules. Die Naturwissenschaften, 58(10), 465-523. doi:10.1007/bf00623322Lynch, M. (2011). The Lower Bound to the Evolution of Mutation Rates. Genome Biology and Evolution, 3, 1107-1118. doi:10.1093/gbe/evr066Bradwell, K., Combe, M., Domingo-Calap, P., & Sanjuán, R. (2013). Correlation Between Mutation Rate and Genome Size in Riboviruses: Mutation Rate of Bacteriophage Qβ. Genetics, 195(1), 243-251. doi:10.1534/genetics.113.154963Drake, J. W. (1991). A constant rate of spontaneous mutation in DNA-based microbes. Proceedings of the National Academy of Sciences, 88(16), 7160-7164. doi:10.1073/pnas.88.16.7160Schmitt, M. W., Kennedy, S. R., Salk, J. J., Fox, E. J., Hiatt, J. B., & Loeb, L. A. (2012). Detection of ultra-rare mutations by next-generation sequencing. Proceedings of the National Academy of Sciences, 109(36), 14508-14513. doi:10.1073/pnas.120871510

Identification of Hammerhead Ribozymes in All Domains of Life Reveals Novel Structural Variations

Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of “type II” hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred

Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs

[EN] Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes.This work was supported by the Ministerio de Economia y Competitividad (grant number BFU2011-23398).Cervera Olagüe, A.; La Peña Del Rivero, MD. (2014). Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs. Molecular Biology and Evolution. 31(11):2941-2947. https://doi.org/10.1093/molbev/msu232S29412947311

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field