Probing <scp>RNA</scp> Structure and Metal‐Binding Sites Using Terbium(<scp>III</scp>) Footprinting

Harris, D. Ahmasi; Walter, Nils G.

doi:10.1002/0471142700.nc0608s13

Cited by 21 publications

(41 citation statements)

References 13 publications

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“…The relative cleavage intensity at the guanine residues is calculated according to Harris and Walter (2003). The normalized cleavage intensities were plotted against Mg 2+ concentration, and the data points were fitted to a 1:1 binding model (Sigel et al 2000) to derive the dissociation constant K D .…”

Section: Evaluation Of Data and Calculation Of K Amentioning

confidence: 99%

Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Choudhary

Sigel

2013

RNA

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Mg2+ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B 12 (adenosylcobalamine, AdoCbl), and down-regulates the expression of the B 12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg 2+ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg 2+ . With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg 2+ (or low Mg 2+ concentration) and the other appearing above ∼0.5 mM Mg 2+ . Distinct regions of the riboswitch exhibit different dissociation constants toward Mg 2+ , indicating a stepwise folding of the btuB RNA. Increasing the Mg 2+ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg 2+ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B 12 , and switch the RNA conformation. Moreover, raising the Mg 2+ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg 2+ indicates a crucial role played by Mg 2+ for defining an active conformation of the riboswitch.

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Section: Evaluation Of Data and Calculation Of K Amentioning

confidence: 99%

Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Choudhary

Sigel

2013

RNA

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“…As our data are consistent with the involvement of divalent cations in catalysis of the snRNA-mediated splicing, we asked whether we could determine the existence and location of sitespecifically bound divalent cations in the U6/U2 complex used in our assays. To this end, we performed terbium(III) cleavage assays (Harris and Walter, 2003;Sigel and Pyle 2003). When present in the reaction mixture at very low concentrations, the sites of cleavage by terbium(III) correspond to site-specific, inner sphere metal binding pockets (Harris and Walter 2003;Sigel and Pyle 2003).…”

Section: Metal Binding By the U6 Isl Acagaga And Agc Sequencesmentioning

confidence: 99%

“…To this end, we performed terbium(III) cleavage assays (Harris and Walter, 2003;Sigel and Pyle 2003). When present in the reaction mixture at very low concentrations, the sites of cleavage by terbium(III) correspond to site-specific, inner sphere metal binding pockets (Harris and Walter 2003;Sigel and Pyle 2003). In these studies, we used a chimeric U6/U2 complex in which the 39 end of U6 was joined to the 59 end of U2 via a UUCG hairpin (Fig.…”

Section: Metal Binding By the U6 Isl Acagaga And Agc Sequencesmentioning

confidence: 99%

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

Lee¹,

Jaladat²,

Mohammadi³

et al. 2010

RNA

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We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 59 and 39 splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.

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“…One micromole of cold CPEB3 RNA was added to reduce the extent of nonspecific RNA degradation by Tb 3+ and to facilitate RNA precipitation (Harris and Walter 2003). For annealing, the RNA was heated to 90°C for 90 s, then transferred to ice and 5 or 20 mM MgCl 2 was added immediately.…”

Section: +mentioning

confidence: 99%

“…The RNA fragments were separated by 16% denaturing PAGE and visualized on a phosphorimager. On each gel, an alkaline hydrolysis ladder (OH − ) and a T1-ribonuclease cleavage ladder of CPEB3 were loaded (Harris and Walter 2003). The OH − ladder was prepared by incubating the same amount of radiolabeled CPEB3 as used in the Tb 3+ -induced cleavage reactions in 50 mM sodium carbonate buffer (pH 9) and 1 mM EDTA for 4-5 min at 90°C and by quenching with formamide loading buffer (1:1).…”

Section: +mentioning

confidence: 99%

Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme

Skilandat

Rowińska‐Żyrek

Sigel

2016

RNA

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Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg 2+ , the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb 3+ -induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg 2+ ions. Up to eight Mg 2+ ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg 2+ promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg 2+ binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg 2+ for the nested double pseudoknot structure.

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Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting

Cited by 21 publications

References 13 publications

Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme

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Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting

Cited by 21 publications

References 13 publications

Mg2+-induced conformational changes in the btuB riboswitch from E. coli

Mg2+-induced conformational changes in the btuB riboswitch from E. coli

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme

Contact Info

Product

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Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Mg²⁺-induced conformational changes in the btuB riboswitch from E. coli

Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme