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Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N -methyl amino acids, D -amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.

The discovery of ribozymes triggered a huge interest in the chemistry and biology of RNAs. Much of the recently made progress focusing on metal ions is addressed in Volume 9. This book, written by 28 internationally recognized experts, provides a most up-to-date view and it is thus of special relevance for colleagues teaching courses in biological inorganic chemistry and for researchers dealing, e.g., with nucleic acids, gene expression, and enzymology, but also for those in analytical and bioinorganic chemistry or biophysics. Structural and Catalytic Roles of Metal Ions in RNA describes metal ion-binding motives, methods to detect and characterize metal ion binding sites, and the role of metal ions in folding and catalysis. It deals with diffuse metal ion binding, RNA quadruplexes, the regulation of riboswitches, metal ions and ribozymes, including artificial ribozymes. The ribosome, ribozymes and redox cofactors, as well as the binding of kinetically inert metal ions to RNA are also considered.

Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. Here we report developing a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identify new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provide conserved sequence and structure motifs that may be used for rationally guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems. Synthetic RNA-based devices can dynamically control a wide range of processes. Here the authors develop a quantitative and high-throughput mammalian cell-based RNA-seq assay to efficiently engineer ribozyme switches.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.

Bacterial group II introns encode maturase proteins required for splicing. In organelles of photosynthetic land plants, most of the group II introns have lost the reading frames for maturases. Here, we show that the plastidial maturase MatK not only interacts with its encoding intron within trnk-UUU, but also with six additional group II introns, all belonging to intron subclass llA. Mapping analyses of RNA binding sites revealed MatK to recognize multiple regions within the trnK intron. Organellar group ll introns are considered to be the ancestors of nuclear spliceosomal introns. That MatK associates with multiple intron ligands makes it an attractive model for an early trans-acting nuclear splicing activity.

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.

It has been suggested that RNA polymerase ribozyme displaying reverse transcriptase and integrase activities has played a vital role in the origin of life on Earth. Here, we present a hypothesis that formation of universal ancestral units of all living organisms — retroelements — in the evolution was mediated by reverse transcriptase. The propensity of retroelements to mutations and their insertion capacity have formed a basis for the origin of complex DNA structures — primary genomes — that have given rise to archaea, eukaryotes, bacteria, and viruses. Conserved properties of retroelements have been preserved throughout the evolution; their modifications have facilitated the emergence of mechanisms for the interactions between proteins and nucleic acids. Life has evolved due to insertional mutagenesis and competition of autonomously replicating polynucleotides that allowed to preserve structures with adaptive properties. We hypothesize that natural selection of mechanisms for the defense against insertions based on the ribonuclease activity of reverse transcriptase ribozyme has led to the emergence of all universal enzymatic systems for the processing of RNA molecules. These systems have been and still remain the key sources of structural and functional transformations of genomes in the course of evolution. The data presented in this review suggest that the process of translation, which unifies the nucleic acid and protein worlds, has developed as a modification of the defense mechanisms against insertions. Polypeptides formed by this defense system have potentiated the activity of ribozymes in the composition of ribonucleoproteins (RNPs) and even functionally replaced them as more efficient catalysts of biological reactions. Here, we analyze the mechanisms of retroelement involvement in the structural and regulatory transformations of eukaryotic genomes supposedly reflecting the adaptive principles that had originated during the beginning of life on Earth. Simultaneously with the evolution of existing proteins, retroelements have served as sources of new ribozymes, such as long non-coding RNAs. These ribozymes can function in complexes with proteins in the composition of RNPs, as well as display independent catalytic and translational activities; their genes have a potential for the transformation into protein-coding genes. Hence, the conserved principles of RNA, DNA, and proteins interregulation formed at the time of life origin on Earth have been used throughout the evolution.

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5' end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.