Probing RNA Structure Within Living Cells

Liebeg, Andreas; Waldsich, Christina

doi:10.1016/s0076-6879(09)68011-3

Cited by 19 publications

(26 citation statements)

References 38 publications

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“…Primer extension and PAGE DMS modifications were assayed by primer extension with radiolabeled oligonucleotides as described in Liebeg and Waldsich (2009) with the following changes. For each reaction, 2.5 μL (500 ng) of whole-cell RNA was incubated with 1 μL (0.2 μM) of 32 P-labeled primer (5 ′ -AAATGACGCTCAAACAGGCATGC-3 ′ ), and 1 μL of 4.5X hybridization buffer (225 mM Hepes at pH 7.0; 450 mM KCl) at 95°C for 5 min.…”

Section: Growth Of Yeast Strains Dms Probing and Rna Extractionmentioning

confidence: 99%

Mod-seq: high-throughput sequencing for chemical probing of RNA structure

Talkish

May

Lin

et al. 2014

RNA

172

212

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The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent-accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method (Mod-seq) and data analysis pipeline (Mod-seeker) for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional noncoding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its highthroughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.

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Section: Growth Of Yeast Strains Dms Probing and Rna Extractionmentioning

confidence: 99%

Mod-seq: high-throughput sequencing for chemical probing of RNA structure

Talkish

May

Lin

et al. 2014

RNA

172

212

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“…The site of modification by Kmr was determined by reverse transcription (RT) primer extension (15) of 16S rRNA from both in vitro and in vivo modified 30S subunits. For in vivo modified 16S rRNA, total rRNA was extracted from E. coli BL21(DE3) cells expressing recombinant Kmr as previously described (16). For in vitro modified 16S rRNA, the RNA was obtained by phenol extraction and ethanol precipitation from purified 30S subunits (E. coli MRE600) following an in vitro methylation reaction with recombinant Kmr.…”

Section: Methodsmentioning

confidence: 99%

30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

Savic

Sunita

Zelinskaya

et al. 2015

Antimicrob Agents Chemother

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bMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core ␤-strands 6 and 7, which forms part of the S-adenosyl-L-methionine (SAM) binding pocket and contributes to base flipping by the m 1 A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. Gene pools in soil and aquatic microbial communities represent largely unexplored reservoirs of antibiotic resistance (1, 2). An analysis of the resistance mechanisms present in these communities may reveal new insights into the origins, activities, and potential for mobilization of antibiotic resistance determinants to pathogenic bacterial populations. In particular, myxobacteria of the genus Sorangium have become a major source of secondary metabolites over the last several decades alongside the actinobacteria and fungi (3, 4). The model strain Sorangium cellulosum So ce56 possesses a 13.1-Mb genome with 17 gene clusters for secondary metabolites (5). Associated with this tremendous biosynthetic power is a high resistance potential against multiple antibiotics. For example, all Sorangium strains can grow in the presence of exceptionally high concentrations of kanamycin through the action of a single, specific aminoglycoside resistance-conferring 16S rRNA methyltransferase, Kmr (6).In common with most aminoglycosides, kanamycin binds within helix (h) 44 of the 16S rRNA in the bacterial small (30S) ribosomal subunit and induces errors in mRNA decoding (7-9). Aminoglycoside-producing bacteria typically protect themselves from self-intoxication by expression of aminoglycoside resistance-conferring methyltransferases that modify the drug binding site. Two subfamilies of 16S rRNA methyltransferases introduce distinct 16S rRNA modifications at N7 of guanosine 1405 (m 7 G1405) or N1 of adenosine 1408 (m 1 A140...

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“…To date, several chemical reagents, which are sensitive to secondary and/or tertiary structure, have been used for probing RNA structure in vivo (reviewed in ref. [118][119][120][121][122]. DMS is the most successfully used chemical to probe RNA structure in a variety of organisms, ranging from bacteria to eukaryotes.…”

Section: Current Methods Employed To Study Rna Structure In Vivomentioning

confidence: 99%

“…DMS is the most successfully used chemical to probe RNA structure in a variety of organisms, ranging from bacteria to eukaryotes. 120,[123][124][125][126][127][128][129][130][131] A major convenience of DMS is that it rapidly penetrates all compartments of cells without prior cell permeabilization. After uptake, DMS methylates N7 of guanines, N1 of adenines and N3 of cytosines, if they are not involved in H-bonding or protected by proteins.…”

Section: Current Methods Employed To Study Rna Structure In Vivomentioning

confidence: 99%