RNA Crosslinking Methods

Harris, Michael E.; Christian, Eric L.

doi:10.1016/s0076-6879(09)68007-1

Cited by 47 publications

(57 citation statements)

References 61 publications

(89 reference statements)

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“…The cross-linked species were excised and analyzed by reverse transcriptase (RTase). RTase is blocked 1 nt upstream from an RNA cross-link (23), which allowed us to locate potential cross-linking sites. Stem I 101 and stem I 86 had two clear RTase blockages, a strong site at G55 and a weak one at C43 (Fig.…”

Section: Selective 2′-hydroxyl Acylation Analyzed By Primer Extensionmentioning

confidence: 99%

T box RNA decodes both the information content and geometry of tRNA to affect gene expression

Grigg¹,

Chen

Grundy

et al. 2013

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

125

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The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven looploop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for basestacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D-and T-loops. Strikingly, the binding platform strongly resembles the D-and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.riboswitch | RNA-RNA complex C ells must maintain appropriate intracellular pools of aminoacylated transfer RNA (aa-tRNA) to survive. This is usually accomplished by tight regulation of many cellular factors, including amino acid biosynthesis and transporter genes, and tRNA synthetases (1). Most Gram-positive bacteria use the T box regulatory system to control their aa-tRNA levels (2). T box elements are a special family of regulatory RNAs located in the 5′-untranslated region of the mRNA for a regulated gene or operon. Unlike small molecule-sensing riboswitches, T box RNAs recognize tRNAs containing the cognate anticodon and discriminate between uncharged tRNA and aa-tRNA. They usually control gene expression via premature transcription termination, although there are rare examples that are predicted to regulate at the level of translation initiation (2-4). The T box motif was initially identified in the Bacillus subtilis (Bsub) tyrosyl-tRNA synthetase gene (tyrS) and was shown to respond to the aminoacylation state of tyrosine tRNA (5, 6). More than 1,000 T box elements have since been identified (3, 4).Selective tRNA recognition is achieved through a collective effort from several conserved domains in the T box RNA [Geobacillus kaustophilus (Gkau) glyQS T box sequence is shown in Fig. 1]. Stem I is the largest element. It is an ∼100-nucleotide (nt) stem-loop s...

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Section: Selective 2′-hydroxyl Acylation Analyzed By Primer Extensionmentioning

confidence: 99%

T box RNA decodes both the information content and geometry of tRNA to affect gene expression

Grigg¹,

Chen

Grundy

et al. 2013

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

125

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“…It should be noted that UV irradiation and functional reagents can also induce protein-protein and RNA-RNA crosslinking; these aspects will not be discussed here, and the interested reader is referred to the relevant literature (e.g. [66,67]). …”

Section: Samples Applied To Uv Crosslinkingmentioning

confidence: 99%

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Schmidt

Kramer

Urlaub

2012

Journal of Proteomics

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“…To increase the efficiency of the cross-linking to the levels that would make a reliable detection of transient enzyme–substrate complexes possible, we substituted uridines in our substrate with 4-thiouridines. The 4-thiouridine is structurally similar to uridine, the only difference being a single atom (oxygen for sulfur) substitution that has a minimal effect on the substrate’s properties but allows for a dramatically increased efficiency of cross-linking on photoactivation (54). The substitution of uridines with 4-thiouridines did not interfere with RNase MRP cleavage (Figure 2); all uridines were substituted with 4-thiouridines during in vitro transcription in the presence of 4-thiouridine and no uridine (‘Materials and Methods’ section).…”

Section: Resultsmentioning

confidence: 99%

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate

Esakova

Perederina

Berezin

et al. 2013

Nucleic Acids Research

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Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

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RNA Crosslinking Methods

Cited by 47 publications

References 61 publications

T box RNA decodes both the information content and geometry of tRNA to affect gene expression

T box RNA decodes both the information content and geometry of tRNA to affect gene expression

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate

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