Combined smFRET and NMR analysis of riboswitch structural dynamics

Bains, Jasleen Kaur; Blechar, Julius; Jesus, Vanessa de; Meiser, Nathalie; Zetzsche, Heidi; Fürtig, Boris; Schwalbe, Harald; Hengesbach, Martin

doi:10.1016/j.ymeth.2018.10.004

Cited by 9 publications

(10 citation statements)

References 46 publications

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“…The 13 C– 15 N-labeled (rATP, rCTP, rGTP, rUTP) nucleotides for transcription were purchased from Silantes (Munich, Germany). The construct was purified by preparative polyacrylamide gel electrophoresis according to standard protocols ( 48 ). The construct was folded in water for 5 min at 95°C and immediately diluted 10-fold with ice-cold water.…”

Section: Methodsmentioning

confidence: 99%

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

Biedenbänder

Jesus

Schmidt

et al. 2022

Nucleic Acids Research

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A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.

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Section: Methodsmentioning

confidence: 99%

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

Biedenbänder

Jesus

Schmidt

et al. 2022

Nucleic Acids Research

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“…RNA preparation : All RNAs were prepared in house through in vitro transcription with T7 RNAP [69] . DNA templates included the necessary T7 promotor and were either obtained from linearizing plasmid containing the sequence of interest or PCR run‐off.…”

Section: Methodsmentioning

confidence: 99%

“…RNA preparation: All RNAs were prepared in house through in vitro transcription with T7 RNAP. [69] DNA templates included the necessary T7 promotor and were either obtained from linearizing plasmid containing the sequence of interest or PCR run-off. Transcription conditions were optimized for yield and sample purity and in vitro transcription was performed in 10 to 20 mL scale dependent on the expected yield.…”

Section: Methodsmentioning

confidence: 99%

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¹⁹F NMR‐Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter‐Screens

et al. 2020

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We report here on the nuclear magnetic resonance (NMR) 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter‐screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow‐up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low micromolar binding affinity without losing binding specificity between two different terminator structures.

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“…The 13 C-15 N-labeled (rATP, rCTP, rGTP, rUTP) nucleotides for transcription were purchased from Silantes (Munich, Germany). tRNA Ile was purified by preparative polyacrylamide gel electrophoresis according to standard protocols (Bains et al 2019). The tRNA Ile was folded in water by heat denaturation for 5 min at 95 °C and immediately diluted tenfold with ice-cold water.…”

Section: Sample Preparationmentioning

confidence: 99%

NMR assignment of non-modified tRNAIle from Escherichia coli

et al. 2022

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AbstracttRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

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Combined smFRET and NMR analysis of riboswitch structural dynamics

Cited by 9 publications

References 46 publications

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

¹⁹F NMR‐Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter‐Screens

NMR assignment of non-modified tRNAIle from Escherichia coli

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Combined smFRET and NMR analysis of riboswitch structural dynamics

Cited by 9 publications

References 46 publications

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

19F NMR‐Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter‐Screens

NMR assignment of non-modified tRNAIle from Escherichia coli

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¹⁹F NMR‐Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter‐Screens