Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.     

Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction

Even if template sequence of hTR played an essential role in telomere binding, a 326 nucleotide fragment of hTR containing template, pseudoknot, and CR4-5 domains is critical for both binding with telomeric DNA and reconstitution of telomerase activity. A functional study with antisense oligonucleotides suggested that targeted disruption of the template region efficiently abrogated both telomeric DNA binding and telomerase activity, whereas disruption of the CR4-5 region induced only loss of telomerase activity. hTR interacts with telomeric DNA via structural region composed of the template, pseudoknot, and CR4-5 domains, however, each structural domain plays a distinct role in telomere binding and telomerase activity reconstitution.     

Hammerhead ribozymes to modulate telomerase activity of endometrial carcinoma cells.

Telomerase is an excellent target molecule for cancer therapy, though any effective agents have never been developed in human subjects. We designed a variety of hammerhead ribozymes against human telomerase RNA (hTR) and hTERT mRNA and studied their possibility as a tool for cancer therapy. To search promising target site of hTR, the catalytic actiuity of 3 kinds of hammerhead ribozymes was studied in cell-free system. They showed equivalent catalytic activity, but only 36-ribozyme, which was designed to cleave the template region of hTR, revealed telomerase inhibitory activity in an endometrial carcinoma cell line. Among hTERT-mRNA-targeted ribozymes, the ribozyme to cleave 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to cleave 59 nucleotides upstream from the poly(A) tail showed clear activity. Stable transfection studies confirmed that the 36-ribozyme as well as the 13-ribozyme suppressed telomerase. These observations suggest that the template region of hTR and 5'end of hTERT mRNA are promising target sites for ribozymes to reduce telomerase activity.     

人端粒酶RNA基因的克隆与鉴定

以人血基因组ＤＮＡ为模板,合成两段２０个寡聚核苷酸为引物,经过ＰＣＲ扩增,得到４８０ｂｐ的片段,克隆到ｐＭＤ１８－Ｔ载体中,经电泳、酶切、ＰＣＲ鉴定后测定序列。序列分析表明氙克隆的人端粒酶ＲＮＡ（ｈｕｍａｎ ｔｅｌｏｍｅａｓｅ ＲＮＡ,ｈＴＲ）基因含有４８０ｂｐ,包括约４５０ｂｐ的编码模板区主序列和约３０ｂｐ的上游调控区序列,其中模板区的１１个核苷酸（５’－ＣＵＡＡＣＣＣＵＡＡＣ－３’）合成端粒亚     

Biochemical analysis of pistol self-cleaving ribozymes

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min⁻¹ under physiological Mg²⁺ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2′-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.     

Biphasic folding kinetics of RNA pseudoknots and telomerase RNA activity

Using a combined master equation and kinetic cluster approach, we investigate RNA pseudoknot folding and unfolding kinetics. The energetic parameters are computed from a recently developed Vfold model for RNA secondary structure and pseudoknot folding thermodynamics. The folding kinetics theory is based on the complete conformational ensemble, including all the native-like and non-native states. The predicted folding and unfolding pathways, activation barriers, Arrhenius plots, and rate-limiting steps lead to several findings. First, for the PK5 pseudoknot, a misfolded 5' hairpin emerges as a stable kinetic trap in the folding process, and the detrapping from this misfolded state is the rate-limiting step for the overall folding process. The calculated rate constant and activation barrier agree well with the experimental data. Second, as an application of the model, we investigate the kinetic folding pathways for human telomerase RNA (hTR) pseudoknot. The predicted folding and unfolding pathways not only support the proposed role of conformational switch between hairpin and pseudoknot in hTR activity, but also reveal molecular mechanism for the conformational switch. Furthermore, for an experimentally studied hTR mutation, whose hairpin intermediate is destabilized, the model predicts a long-lived transient hairpin structure, and the switch between the transient hairpin intermediate and the native pseudoknot may be responsible for the observed hTR activity. Such finding would help resolve the apparent contradiction between the observed hTR activity and the absence of a stable hairpin.     

Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.     

Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure

The glmS ribozyme is a self-cleaving RNA catalyst that resides in the 5′-untranslated region of glmS mRNA in certain bacteria. The ribozyme is specifically activated by glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein, and is thus proposed to provide a feedback mechanism of riboswitch regulation. Both phylogenetic and biochemical analyses of the glmS ribozyme have established a highly conserved core sequence and secondary structure required for GlcN6P-dependent self-cleavage. However, the high degree of nucleotide conservation offers few clues regarding the higher-order structural organization of the catalytic core. To further investigate core ribozyme structure, minimal ‘consensus-type’ glmS ribozymes that retain GlcN6P-dependent activity were produced. Mutational analyses of consensus-type glmS ribozymes support a model for core ribozyme folding through a pseudoknot structure formed by the interaction of two highly conserved sequence segments. Moreover, GlcN6P-dependent function is demonstrated for bimolecular constructs in which substrate interaction with the ribozyme is minimally comprised of sequence representing that involved in putative pseudoknot formation. These studies suggest that the glmS ribozyme adopts an intricate multi-strand catalytic core through the formation of a pseudoknot structure, and provide a refined model for further considering GlcN6P interaction and GlcN6P-dependent ribozyme function.