The kinetics of ribozyme cleavage: a tool to analyze RNA folding as a function of catalysis.

As catalytically active RNAs, ribozymes can be characterized by kinetic measurements similar to classical enzyme kinetics. However, in contrast to standard protein enzymes, for which reactions can usually be started by mixing the enzyme with its substrate, ribozymes are typically self-cleaving. The reaction has to be initiated by folding the RNA into its active conformation.

The low incidence of diversity-generating retroelements in sequenced genomes.

The insertion of a retrotransposable element is usually associated with adverse or, at best, neutral effects on the host. Diversity-generating retroelements (DGRs) are the first elements that seem to offer a direct selective advantage to their phage or prokaryote host by exact replacement of a short, defined region of a host gene with a hypermutated variant. In a previous study, we presented the software DiGReF for identification of DGRs in genome sequences, and compiled the first comprehensive set of diversity-generating retroelements in public databases.

Analysis of a comprehensive dataset of diversity generating retroelements generated by the program DiGReF.

Background: Diversity Generating Retroelements (DGRs) are genetic cassettes that can introduce tremendous diversity into a short, defined region of the genome. They achieve hypermutation through replacement of the variable region with a strongly mutated cDNA copy generated by the element-encoded reverse transcriptase. In contrast to "selfish" retroelements such as group II introns and retrotransposons, DGRs impart an advantage to their host by increasing its adaptive potential.

Dual roles for the Mss116 cofactor during splicing of the ai5γ group II intron.

The autocatalytic group II intron ai5γ from Saccharomyces cerevisiae self-splices under high-salt conditions in vitro, but requires the assistance of the DEAD-box protein Mss116 in vivo and under near-physiological conditions in vitro. Here, we show that Mss116 influences the folding mechanism in several ways. By comparing intron precursor RNAs with long (∼300 nt) and short (∼20 nt) exons, we observe that long exon sequences are a major obstacle for self-splicing in vitro.

Protein-facilitated ribozyme folding and catalysis.

In vivo, large RNAs rely on proteins to fold to their native conformation. In the case of the S. cerevisiae group II intron ai5 gamma, the DEAD-box protein Mss116 has been shown to promote the formation of the catalytically active structure.

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for approximately 28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations.

Group II introns: structure, folding and splicing mechanism.

Group II introns are large autocatalytic RNAs found in organellar genomes of plants and lower eukaryotes, as well as in some bacterial genomes. Interestingly, these ribozymes share characteristic traits with both spliceosomal introns and non-LTR retrotransposons and may have a common evolutionary ancestor. Furthermore, group II intron features such as structure, folding and catalytic mechanism differ considerably from those of other large ribozymes, making group II introns an attractive model system to gain novel insights into RNA biology and biochemistry.

A DEAD protein that activates intron self-splicing without unwinding RNA.

The group II intron ai5gamma from S. cerevisiae requires high temperature and salt to self-splice in vitro, but it is assisted by the protein Mss116 in vivo. Here we show that Mss116 can stimulate splicing of ai5gamma under near-physiological conditions in vitro, which represents one of the first cases in which a DExH/D protein is shown to act on its natural target.

APE-type non-LTR retrotransposons: determinants involved in target site recognition.

Non-long terminal repeat (Non-LTR) retrotransposons represent a diverse and widely distributed group of transposable elements and an almost ubiquitous component of eukaryotic genomes that has a major impact on evolution. Their copy number can range from a few to several million and they often make up a significant fraction of the genomes. The members of the dominating subtype of non-LTR retrotransposons code for an endonuclease with homology to apurinic/apyrimidinic endonucleases (APE), and are thus termed APE-type non-LTR retrotransposons.

Analysis of 5' junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5'-end attachment requiring microhomology-mediated end-joining.

Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3'-end of an L1 copy to the genomic target, but the subsequent integration steps leading to the attachment of the 5'-end to the chromosomal DNA are still cause for speculation. By applying bioinformatics to analyze the 5' junctions of recent L1 insertions in the human genome, we provide evidence that L1 uses at least two distinct mechanisms to link the 5'-end of the nascent L1 copy to its genomic target.

Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription.

In order to explore the defense mechanism by which retrotransposons are repressed, we assessed the ability of methyl-CpG-binding protein 2, MeCP2, to influence LINE-1 (L1) and Alu transcription and, furthermore, L1 retrotransposition. In transient transfection assays, targeting of the transcriptional-repression domain (TRD) of MeCP2 (via a linked Gal4 DNA-binding domain) to the transcriptional start site of L1 promoter-driven reporter constructs efficiently repressed transcription. The Gal4-linked TRD of the related methyl-CpG-binding protein MBD1 also repressed transcription but not that of MBD2.

Domain structure of riboflavin synthase.

Riboflavin synthase of Escherichia coli is a homotrimer of 23.4 kDa subunits catalyzing the formation of the carbocyclic ring of the vitamin, riboflavin, by dismutation of 6,7-dimethyl-8-ribityllumazine. Intramolecular sequence similarity suggested that each subunit folds into two topologically similar domains.