Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.
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PMID:Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group. 1049 33

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.
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PMID:Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. 1100 93

Due to trans-splicing and polycistronic transcription, the 5' end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5' ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5' ends. In contrast, pol II primary transcripts, the 5' triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5' end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5' end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5' end of RNA transcripts was characterized. We found that: (1). the 5' end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2). approximately 20% of the putative rRNA precursor contained a 5' tri- or diphosphate group, representing the primary transcript and approximately 80% of the putative rRNA precursor were dephosphorylated and contained a 5' hydroxyl group; (3). the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5' hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5' tri- or diphosphate, was below a detectable level in the steady state RNA; and (4). we did not detect pol I transcribed precursor RNAs that contained a 5' monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5' capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.
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PMID:The 5' end structure of transcripts derived from the rRNA gene and the RNA polymerase I transcribed protein coding genes in Trypanosoma brucei. 1279 8

A simple strategy is reported for 5'-adenylation of nearly any RNA sequence of indefinite length. The 5'-adenylated product (5'-AppRNA) is an activated RNA that is structurally similar to 5'-triphosphorylated RNA, which is usually prepared by in vitro transcription using T7 RNA polymerase. In the new 5'-adenylation strategy, the RNA substrate is first 5'-monophosphorylated either by T4 polynucleotide kinase, by in vitro transcription in the presence of excess GMP, or by appropriate derivatization during solid-phase synthesis. The RNA is then 5'-adenylated using ATP and T4 RNA ligase, in an interrupted version of the natural adenylation-ligation mechanism by which T4 RNA ligase joins two RNA substrates. Here, the final ligation step of the mechanism is inhibited with complementary DNA blocking oligonucleotide(s) that permit adenylation to occur with good yield. The 5'-AppRNA products of this approach should be valuable as activated RNAs for in vitro selection experiments as an alternative to 5'-triphosphorylated RNAs, among other likely applications. The 5'-terminal nucleotide of an RNA substrate to be adenylated using the new method is not restricted to guanosine, in contrast to 5'-triphosphorylated RNA prepared by in vitro transcription. Therefore, using the new approach, essentially any RNA obtained from solid-phase synthesis or other means can be activated by 5'-adenylation in a practical manner.
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PMID:Practical and general synthesis of 5'-adenylated RNA (5'-AppRNA). 1503 82

The search is underway for a catalytic RNA molecule capable of self-replication. Finding such a ribozyme would lend crucial support to the RNA World hypothesis, which holds that very early life-forms relied on RNA for both replicating and storing genetic information. We previously reported an RNA polymerase isolated from a pool of variants of an existing RNA ligase ribozyme. Here we report eight additional ligase-derived polymerase ribozymes isolated from this pool. Because each of them is a new potential starting point for further in vitro evolution and engineering, together they substantially enrich the set of candidates from which an RNA replicase ribozyme might eventually emerge.
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PMID:New ligase-derived RNA polymerase ribozymes. 1598 4

The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5' termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5'-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels.
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PMID:A short double-stranded RNA motif of Peach latent mosaic viroid contains the initiation and the self-cleavage sites of both polarity strands. 1618 95

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1alpha to activate its protein kinase and endoribonuclease activities. Activated IRE1alpha cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5' and 3' fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1alpha and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F(v)-IRE1alphaDeltaN was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1alpha dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.
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PMID:Cytoplasmic IRE1alpha-mediated XBP1 mRNA splicing in the absence of nuclear processing and endoplasmic reticulum stress. 1664 24

We have cloned a ZRT (Zn-regulated transporter), IRT (Fe-regulated transporter)-like protein (ZIP) gene from the strawberry plant (Fragaria x ananassa Duch). This gene, designated as FaZIP1, is composed of three exons and two introns. The locations of the two introns (546 and 157 base pairs) in the gene were confirmed by sequence analysis of cDNA clones obtained by using 3' and 5' RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) PCR. Also, based on the cDNA sequence, the transcriptional start site of FaZIP1 was assigned. FaZIP1 is predicted to code for a protein of 353 amino acids containing a signal peptide and eight potential transmembrane domains. Southern blot analysis indicated that FaZIP1 is a member of a multi-gene family. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the FaZIP1 gene is constitutively expressed in strawberry leaves and roots.
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PMID:Identification of a zinc transporter gene in strawberry. 1675 13

A full-length cloned cDNA insert containing the sequence of potato spindle tuber viroid (PSTV) was dimerized and placed in the plasmid vector pSP65 adjacent to a promoter for bacteriophage SP6 RNA polymerase. In vitro transcription of this region yielded a single linear RNA chain 760 bases in length containing two copies of PSTV RNA with about 20 bases of vector sequence at each end. Bioassay on tomato plants revealed that this transcript has infectivity comparable to that of PSTV itself, yielding circular progeny RNAs indistinguishable from PSTV grown in vivo Comparative RNA fingerprinting analysis revealed that the viroid sequence in the dimeric transcript breeds true (compared to control viroid strains of different sequence grown in parallel) but loses the vector-specific sequences during growth in plants. Incubation of 32P-labeled dimeric PSTV RNA at neutral pH and 39 degrees gave a 1-5% yield of three RNA segments, one comigrating with unit-length linear PSTV and two smaller fragments. Reaction of the unit-length cleavage product with wheat germ RNA ligase gave a high yield of circular molecules indistinguishable from PSTV circles arising in vivo, suggesting that the cleavage reaction yields 2',3' cyclic phosphate termini and could represent a part of the in vivo PSTV replication cycle.
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PMID:Cell-free synthesis and processing of an infectious dimeric transcript of potato spindle tuber viroid RNA. 1863 47

Ribozyme-catalyzed RNA synthesis is central to the RNA world hypothesis. No natural RNA polymerase ribozymes have been discovered. However, ribozymes that catalyze the requisite chemistry, generating a new phosphodiester through attack of a terminal hydroxyl of an RNA on the alpha-phosphate of a triphosphate-activated oligonucleotide, have been isolated by in vitro selection. These experiments often yield ribozymes that generate 2'-5' phosphodiesters rather than conventional 3'-5' linkages. We have determined crystal structures of the duplex formed by the template segment of a representative 2'-5' RNA ligase ribozyme, the class II ligase, and its ligation product. The structures reveal a product-template duplex with a G x A pair at the ligation junction. This sheared pair is flanked on one side by a minor groove-broadening wedge comprised of two unpaired nucleotides. The reported structure of an independently isolated 3'-5' ligase ribozyme, the L1 ligase, shows a product-template duplex that shares the G x A pair with the class II ligase. However, this G x A pair is flanked by G x U wobbles, rather than an unpaired wedge. We demonstrate that these structural differences of the substrate-template duplexes are largely responsible for the divergent regioselectivity of the two ribozymes, independent of their catalytic moieties, by constructing chimeras. The L1 ligase with a class II substrate-template duplex shows a 30-fold increase in 2'-5' bond synthesis, while the class II ligase with an L1 substrate-template duplex produces 3'-5' bonds exclusively. These results demonstrate how local geometry inherent to the substrate-template duplexes controls the regioselectivity of ribozyme-catalyzed RNA ligation reactions.
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PMID:Structure-guided engineering of the regioselectivity of RNA ligase ribozymes. 1922 54


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