Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria

BACKGROUND: Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group. RESULTS: We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other α-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds. CONCLUSION: SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups

Entwicklung eines Twinribozyms für die RNA-Reparatur

Ziel dieser Arbeit war die Entwicklung eines Twinribozyms, das durch zweifache Spaltung und anschließende zweifache Ligation den Fragmentaustausch innerhalb einer RNA-Substratsequenz katalysiert. Als Basis für dieses Konzept diente das Hairpinribozym, das durch die Einführung einer zusätzlichen Helix derart verändert wurde, dass die Verknüpfung zweier Einheiten in einem Twinribozym möglich wurde. Weitere Sequenz- und Strukturoptimierung führte zu zwei Einzelmotiven mit hoher Substratselektivität und verbesserter Kofaktorakzeptanz. Durch die Verwendung von 5-Benzylmercapto-1H-tetrazol als Aktivator beim Standard-Phosphoramidit-Verfahren konnte die chemische RNA-Synthese in einem Umfang verbessert werden, der die Synthese eines Twinribozyms mit einer Länge von 141 Nukleotiden und beidseitiger Spaltaktivität erlaubte. Auf enzymatischem Weg wurde ein Twinribozym HP-TW5 synthetisiert, das aus einem 45-mer RNA-Substrat ein 16-mer Fragment herausschneidet und durch ein 20-mer Reparaturfragment ersetzt. Das nach Ligation in bis zu 30 % Ausbeute entstandene 49-mer Produkt wurde durch Sequenzanalyse eindeutig nachgewiesen. Auf diese Weise konnte die Reparatur einer Deletionsmutation in einer Modellreaktion erfolgreich durchgeführt werden. Die Twinribozym-vermittelte RNA-Reparatur bietet damit einen neuartigen Zugang zu gentherapeutischen Anwendungen.Aim of this work was the development of a twin ribozyme that catalyses the fragment exchange within a RNA substrate sequence by twofold cleavage and subsequent twofold ligation. The hairpin ribozyme, serving as basis for this concept, was changed by introduction of an additional helix in a way, that connection of two units in a twin ribozyme became possible. Further sequence and structure optimization led to two different motifs with high substrate selectivity and improved activity under different cofactor conditions. By application of 5-Benzylmercapto-1H-tetrazole as activator in the standard phosphoramidite method, chemical RNA synthesis could be highly improved, allowing synthesis of a Twinribozyme with a length of 141 nucleotides and cleavage activity of both units. The enzymatically synthesised twin ribozyme HP-TW5 was used to cut out a 16-mer fragment from a 45-mer RNA substrate and replace it with a 20-mer repair fragment. The chemical identity of the 49-mer repair product, resulting from ligation in up to 30 percent yield, was proven by sequence analysis. With this model reaction, the repair of a deletion mutation could be successfully demonstrated. Twin ribozyme-mediated RNA-repair offers access to new applications in gene therapy

Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis

Most riboswitches are composed of a single metabolite-binding aptamer and a single expression platform that function together to regulate genes in response to changing metabolite concentrations. In rare instances, two aptamers or sometimes two complete riboswitches reside adjacent to each other in untranslated regions (UTRs) of mRNAs. We have examined an example of a tandem riboswitch in the Gram-positive bacterium Bacillus anthracis that includes two complete riboswitches for thiamine pyrophosphate (TPP). Unlike other complex riboswitch systems described recently, tandem TPP riboswitches do not exhibit cooperative ligand binding and do not detect two different types of metabolites. In contrast, both riboswitches respond independently to TPP and are predicted to function in concert to mimic the more “digital” gene control outcome observed when two aptamers bind ligands cooperatively. Our findings further demonstrate that simple gene control elements made only of RNA can be assembled in different architectures to yield more complex gene control outcomes