The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes

Idiosyncratic cleavage and ligation activity of individual hammerhead ribozymes and core sequence variants thereof

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich. - This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively

Characterization of hammerhead ribozyme reactions

Hammerhead ribozymes are small catalytic RNA motifs ubiquitously present in a large variety of genomes. The reactions catalyzed by these motifs are both their self-scission and the reverse ligation reaction. Here, we describe methods for the generation of DNA templates for the subsequent in vitro transcription of hammerhead ribozymes. This is followed by a description of the preparation of suitable RNA molecules for both reaction types, and their kinetic analysis

Functional hammerhead ribozymes naturally encoded in the genome of Arabidopsis thaliana.

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg(2+) concentrations, indicative of functional loop-loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function

Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg(2+) concentrations, indicative of functional loop–loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function

Ribonucleases in bacterial toxin–antitoxin systems

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNAfMet in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms

ΦTE morphology and genome overview.

<p>(A–B) Transmission electron micrographs of individual ΦTE virus particles. The tail is fully extended in (A) and contracted in (B). Each scale bar represents 100 nm. (C) Summary of the 142,349 bp circularly-permuted genome of ΦTE, including all ORFs (colour coded to function where possible), two tRNAs and the ncRNA comprising pseudo-ToxI, encoded by the escape locus (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003023#pgen.1003023.s003" target="_blank">Table S2</a>). Selected ΦTE genes are indicated by “TE_x” around the genome, for orientation. GC skew and GC content are shown along with the tBLASTx results against two related phages, coliphage rv5 and <i>Salmonella</i> phage PVP-SE1.</p

An excess of pseudo-ToxI inhibits abortive infection.

<p>(A) Strains of Pba ToxIN (pTRB101) were tested for their ability to abort infection in the presence of a second, pBluescript II SK- based, antitoxic plasmid. Putatively antitoxic “test RNA” sequences were cloned under the control of the constitutive <i>lacZα</i> promoter, to allow for constant, high-level, expression. (B) EOPs of ΦTE, ΦM1 and ΦS61 on double strains of Pba, as per key, using Pba ToxIN-FS (pTRB102, pBluescript II SK-) as the control strain. Inserts in the second, antitoxic, plasmids are indicated by the horizontal axis labels. Plasmid pBluescript II SK- was used as the no insert, “vector”, control. “ΦTE escape locus” includes the escape locus from wild type ΦTE, whilst the “ΦTE genomic section” is a 269 bp region of the ΦTE genome, taken several kb from the escape locus as a negative control. Error bars indicate the standard deviation of triplicate (minimum) data.</p