In-Line Probing Analysis of Riboswitches

Regulski, Elizabeth E.; Breaker, Ronald R.

doi:10.1007/978-1-59745-033-1_4

Cited by 303 publications

(344 citation statements)

References 10 publications

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“…We therefore introduced a mutation to this region and measured the binding affinity and cooperativity using in-line probing to obtain the apparent K d and Hill coefficient (n), respectively (Table 1; Regulski and Breaker 2008). NAIM experiments showed N-methyl G interference at G127, which suggested that the exocyclic amine of the nucleotide could be involved in minor groove hydrogenbonding interactions (Rife et al 1998;Kwon and Strobel 2008).…”

Section: Resultsmentioning

confidence: 99%

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Erion¹,

Strobel²

2010

RNA

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The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.

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Section: Resultsmentioning

confidence: 99%

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Erion¹,

Strobel²

2010

RNA

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“…Ribonucleases with different cleavage specificities are also used to identify single-or double-stranded regions, as well as protein-protected regions (RNase footprinting). Other methods such as selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) (Wilkinson et al 2006) and in-line probing (Regulski and Breaker 2008) assess local nucleotide flexibility. SHAPE has since been adapted for high-throughput analysis (SHAPE-Seq) (Lucks et al 2011), joining other techniques that rely on RNase digestion such as fragmentation sequencing (FragSeq) (Underwood et al 2010) and parallel analysis of RNA structure (PARS) (Kertesz et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Long Noncoding RNAs: Past, Present, and Future

2013

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Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years as a potentially new and crucial layer of biological regulation. lncRNAs of all kinds have been implicated in a range of developmental processes and diseases, but knowledge of the mechanisms by which they act is still surprisingly limited, and claims that almost the entirety of the mammalian genome is transcribed into functional noncoding transcripts remain controversial. At the same time, a small number of well-studied lncRNAs have given us important clues about the biology of these molecules, and a few key functional and mechanistic themes have begun to emerge, although the robustness of these models and classification schemes remains to be seen. Here, we review the current state of knowledge of the lncRNA field, discussing what is known about the genomic contexts, biological functions, and mechanisms of action of lncRNAs. We also reflect on how the recent interest in lncRNAs is deeply rooted in biology's longstanding concern with the evolution and function of genomes.T HE past several years have witnessed a steep rise of interest in the study of lncRNAs. Almost on a weekly basis, it seems that a new lncRNA is found to be up-or downregulated in a particular disease, or a new class of noncoding transcripts is uncovered by a transcriptomic study, or a new article heralds a paradigm shift that lncRNAs will bring to our understanding of biology. Without a doubt, the advent of sensitive, high-throughput genomic technologies such as microarrays and next-generation sequencing (NGS) has resulted in an unprecedented ability to detect novel transcripts, the vast majority of which seem not to be derived from annotated protein-coding genes. Despite this explosion of data, however, surprisingly little is known about how lncRNAs function, how many different types of lncRNAs exist, or even whether most of them carry biological significance.In this review, we focus on recently discovered lncRNAs of .200 nt, place these new discoveries in historical context, and outline areas where additional work is needed. The review does not cover "classic" ncRNAs such as ribosomal (r) RNAs, ribozymes, transfer (t)RNAs, small nuclear (sn)RNAs, small nucleolar (sno)RNAs, and telomere-associated RNAs (TERC, TERRA); nor does it cover small ncRNAs such as microRNAs (miRNAs), endogenous small interfering (endo-si)RNAs that participate in RNA interference (RNAi), and Piwi-associated (pi)RNAs. We refer readers to the many

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“…In vitro transcribed RNA was radiolabeled with γ-32 P ATP using T4 polynucleotide kinase (New England Biolabs) following standard procedures (38). After PAGE purification, RNAs were passed through an Illustra MicroSpin G-25 Column (GE Healthcare) and eluted with ddH 2 O. Inline probing assays (1× in-line buffer: 50 mM Tris·HCl, pH 8.3, 20 mM MgCl 2 , 100 mM KCl) were performed as previously described (37) for the extended Gs1761 riboswitch with terminator construct.…”

Section: Bioinformatic Analysis Of Gemm-i Variantsmentioning

confidence: 99%

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

Kellenberger¹,

Wilson²,

Hickey³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

120

146

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Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

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In-Line Probing Analysis of Riboswitches

Cited by 303 publications

References 10 publications

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Long Noncoding RNAs: Past, Present, and Future

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

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