Ligand‐ and pH‐Induced Conformational Changes of RNA Domain Helix 69 Revealed by 2‐Aminopurine Fluorescence

Sakakibara, Yogo; Abeysirigunawardena, Sanjaya; Duc, Anne Cécile E; Dremann, Danielle N.; Chow, Christine S.

doi:10.1002/anie.201206000

Cited by 17 publications

(19 citation statements)

References 30 publications

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“…Differing changes in fluorescence were observed upon titration of the aminoglycosides with pseudouridylated H69 containing a 2-aminopurine substitution at position 1913. 40 In that case, neomycin binding had the biggest impact on the H69 loop conformation relative to the other aminoglycosides, and similar increases in fluorescence were observed with Mg 2+ titrations. However, for DEPC probing at A1913, opposite trends were observed with Mg 2+ and neomycin titrations, suggesting different ligand-induced conformational states.…”

Section: Resultssupporting

confidence: 53%

“…A detailed mechanism for the binding and conformational rearrangement induced by neomycin is not known, but the activity is consistent with the observation that only neomycin induces a different H69 loop structure and causes a more open conformation in the smaller model RNA systems. 35,40 Combined with the DMS probing results, it is inferred that the H69 conformational change with concomitant chemical protection is induced by the interaction of neomycin with the H69 stem in 70S ribosomes.…”

Section: Resultsmentioning

confidence: 89%

“…In contrast, the apparent K d value of neomycin for a small hairpin H69 RNA with modified bases (Ψ1911-m 3 Ψ1915-Ψ1917) was 1.1 µM under identical conditions, consistent with previous results. 37,40 Combined with the footprinting analysis, H69 appears to be a secondary binding site for aminoglycoside antibiotics, with a three- to 10-fold difference in affinity between the 30S and 50S sites of the complete ribosome. These results illustrate the general lack of selectivity of aminoglycosides, even at the ribosome level.…”

Section: Resultsmentioning

confidence: 99%

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Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Sakakibara

Chow

2017

Org. Biomol. Chem.

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Development of antibiotics that target new regions of functionality is a possible way to overcome antibiotic resistance. In this study, the interactions of aminoglycoside antibiotics with helix 69 of the E. coli 23S rRNA in the context of complete 70S ribosomes or the isolated 50S subunit were investigated by using chemical probing and footprinting analysis. Helix 69 is a dynamic RNA motif that plays major roles in bacterial ribosome activity. Neomycin, paromomycin, and gentamicin interact with the stem region of helix 69 in complete 70S ribosomes, but have diminished binding to the isolated 50S subunit. Pseudouridine modifications in helix 69 were shown to impact the aminoglycoside interactions. These results suggest a requirement for a specific conformational state of helix 69 for efficient aminoglycoside binding, and imply that this motif may be a suitable target for mechanism-based therapeutics.

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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

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Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Sakakibara

Chow

2017

Org. Biomol. Chem.

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“…It is possible to determine an equilibrium binding constant for DNA and small molecule systems and there are examples in literature demonstrating this. [18, 20] The ability to determine binding constants for dicationic diamidines is primarily limited to the loss of ligand during injection and response factors for the DNA and complexes, and these limitations influence our preference to use ESI-MS for qualitative purposes only.…”

Section: Discussionmentioning

confidence: 99%

Resolution of Mixed Site DNA Complexes with Dimer‐Forming Minor‐Groove Binders by Using Electrospray Ionization Mass Spectrometry: Compound Structure and DNA Sequence Effects

Laughlin

Wang

Kumar

et al. 2015

Chemistry A European J

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Small molecule targeting of the DNA minor groove is a promising approach to modulate genomic processes necessary for normal cellular function. For instance, dicationic diamindines, a well-known class of minor groove binding compounds, have been shown to inhibit interactions of transcription factors binding to genomic DNA. The applications of these compounds could be significantly expanded if we understand sequence-specific recognition of DNA better and could use the information to design more sequence-specific compounds. Aside from polyamides, minor groove binders typically recognize DNA at A-tract or alternating AT base pair sites. Targeting sites with GC base pairs, referred to here as mixed base pair sequences, is much more difficult than those rich in AT base pairs. Compound 1 is the first dicationic diamidine reported to recognize a mixed base pair site. It binds in the minor groove of ATGA sequences as a dimer with positive cooperativity. Due to the well-characterized behavior of 1 with ATGA and AT rich sequences, it provides a paradigm for understanding the elements that are key for recognition of mixed sequence sites. Electrospray ionization mass spectrometry (ESI-MS) is a powerful method to screen DNA complexes formed by analogs of 1 for specific recognition. We also report a novel approach to determine patterns of recognition by 1 for cognate ATGA and ATGA-mutant sequences. We found that functional group modifications and mutating the DNA target site significantly affect binding and stacking, respectively. Both compound conformation and DNA sequence directionality are crucial for recognition.

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“…168 These effects are equally present in human H69 169,170 and in whole ribosomes 171 , and influence ribosomal subunit association. 172 These conformational effects still await full clarification.…”

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confidence: 99%

Pseudouridine: Still mysterious, but never a fake (uridine)!

Spenkuch

Motorin

Helm³

2014

RNA Biology

179

186

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Pseudouridine (C) is the most abundant of >150 nucleoside modifications in RNA. Although C was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of C synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

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Ligand‐ and pH‐Induced Conformational Changes of RNA Domain Helix 69 Revealed by 2‐Aminopurine Fluorescence

Cited by 17 publications

References 30 publications

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Resolution of Mixed Site DNA Complexes with Dimer‐Forming Minor‐Groove Binders by Using Electrospray Ionization Mass Spectrometry: Compound Structure and DNA Sequence Effects

Pseudouridine: Still mysterious, but never a fake (uridine)!

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