Isothermal Titration Calorimetry Measurements of Riboswitch-Ligand Interactions

Jones, Christopher P.; Piszczek, Grzegorz; Ferré-D′Amaré, A.R.

doi:10.1007/978-1-4939-9179-2_6

Cited by 10 publications

(10 citation statements)

References 27 publications

(32 reference statements)

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“…All ITC measurements were performed and analyzed with the following method 51 , using a MicroCal iTC200 (Malvern, Egham, UK). Briefly, RNAs were refolded by heating at 95 °C for 2 min and immediately placed on ice.…”

Section: Methodsmentioning

confidence: 99%

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Hua¹,

Jones

Mitra

et al. 2020

Nat Commun

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RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.

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Section: Methodsmentioning

confidence: 99%

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Hua¹,

Jones

Mitra

et al. 2020

Nat Commun

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“…Data were fitted to a single-site binding model with Origin 7.0 ITC software (Microcal Software) to determine the apparent dissociation constant, K D,app ; representative data and fits are shown in Supplementary Figure S1. Data and statistical analysis was performed in accordance to best practice as described (23,24).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for 2′-deoxyguanosine recognition by the 2′-dG-II class of riboswitches

Matyjasik

Batey

2019

Nucleic Acids Research

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A recent bioinformatic analysis of well-characterized classes of riboswitches uncovered subgroups unable to bind to the regulatory molecule of the parental class. Within the guanine/adenine class, seven groups of RNAs were identified that deviate from the consensus sequence at one or more of three positions directly involved purine nucleobase recognition, one of which was validated as a second class of 2′-deoxyguanosine riboswitch (called 2′-dG-II). To understand how 2′-dG-II riboswitches recognize their cognate ligand and how they differ from a previously identified class of 2′-deoxyguanosine binding riboswitches, we have solved the crystal structure of a 2′-dG-II aptamer domain bound to 2′-deoxyguanosine. This structure reveals a global architecture similar to other members of the purine riboswitch family, but contains key differences within the ligand binding core. Defining the 2′-dG-II riboswitches is a two-nucleotide insertion in the three-way junction that promotes novel base-base interactions. Unlike 2′-dG-I riboswitches, the 2′-dG-II class only requires local changes to the ligand binding pocket of the guanine/adenine class to achieve a change in ligand preference. Notably, members of the 2′-dG-II family have variable ability to discriminate between 2′-deoxyguanosine and riboguanosine, suggesting that a subset of 2′-dG-II riboswitches may bind either molecule to regulate gene expression.

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“…Isothermal Titration Calorimetry ITC measurements were performed using a MicroCal iTC 200 microcalorimeter (Malvern) and data were fit using NITPIC and SEDPHAT as previously described (Jones et al, 2019). All experiments were performed at 37 C in 50 mM Hepes-KOH, pH 7.4, 150 mM KCl, 10 mM MgCl 2 , except for 7 and 13, which were supplemented with 3% DMSO.…”

Section: Star+methodsmentioning

confidence: 99%