Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The reactivity at G530, U531 and A532 to the chemical probes kethoxal, CMCT and DMS respectively was increased in the mutant 16S rRNA compared with the wild-type, suggesting a more open 530 loop structure in the mutant ribosomes. This was supported by oligonucleotide binding experiments in which probes complementary to positions 520-526 and 527-533, but not control probes, showed increased binding to the 517C mutant 70S ribosomes compared with the non-mutant control. Furthermore, enzymatic digestion of 70S ribosomes with RNase T1, specific for single-stranded RNA, substantially cleaved both wild-type and mutant rRNAs between G524 and C525, two of the nucleotides involved in the 530 loop pseudoknot. This site was also cleaved in the 517C mutant, but not wild-type rRNA, by RNase V1. Such a result is still consistent with a more open 530 loop structure in the mutant ribosomes, since RNase V1 can cut at appropriately stacked single-stranded regions of RNA. Together these data indicate that the 517C mutant rRNA has a rather extensively unfolded 530 loop structure. Less extensive structural changes were found in mutants 517A and 517U, which caused less misreading. A correlation between the structural changes in the 530 loop and impaired translational accuracy is proposed.
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PMID:Structural changes in the 530 loop of Escherichia coli 16S rRNA in mutants with impaired translational fidelity. 756 70

DEx(D)/(H) proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx(D)/(H) protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5' of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.
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PMID:Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme. 1517 85

Structure mapping experiments (using probes such as dimethyl sulfate [DMS], kethoxal, and T1 and V1 RNases) are used to determine the secondary structures of RNA molecules. The process is iterative, combining the results of several probes with constrained minimum free-energy calculations to produce a model of the structure. We aim to evaluate whether particular probes provide more structural information, and specifically, how noise in the data affects the predictions. Our approach involves generating "decoy" RNA structures (using the sFold Boltzmann sampling procedure) and evaluating whether we are able to identify the correct structure from this ensemble of structures. We show that with perfect information, we are always able to identify the optimal structure for five RNAs of known structure. We then collected orthogonal structure mapping data (DMS and RNase T1 digest) under several solution conditions using our high-throughput capillary automated footprinting analysis (CAFA) technique on two group I introns of known structure. Analysis of these data reveals the error rates in the data under optimal (low salt) and suboptimal solution conditions (high MgCl(2)). We show that despite these errors, our computational approach is less sensitive to experimental noise than traditional constraint-based structure prediction algorithms. Finally, we propose a novel approach for visualizing the interaction of chemical and enzymatic mapping data with RNA structure. We project the data onto the first two dimensions of a multidimensional scaling of the sFold-generated decoy structures. We are able to directly visualize the structural information content of structure mapping data and reconcile multiple data sets.
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PMID:Evaluation of the information content of RNA structure mapping data for secondary structure prediction. 2041 17

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3'-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation.
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PMID:Expanded CUG repeats in DMPK transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences. 3070 May 78