Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Abstract

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 3 2 bulged RNA and one 2 3 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 3 2 bulge (C65 and G66). The RNA WT construct was among the most stable (DG°3 7 = -7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A d G mismatch of the bulge decreases the stability of the construct~0.5-1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA-Mg 2+ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/ mol relative to 1 M salt. Formation of a single A+ d C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+ d C mismatch base pairs together contribute about 2 kcal/mol.

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

“…The factors contributing to interactions with ions are unclear. We have previously shown differences in stability and ion interactions between RNA and DNA constructs in a 2 × 2 symmetrical internal loop constructs (Furniss and Grover 2011). We are further exploring the factors that contribute to the thermodynamic differences in various nonhelical structures in RNA and DNA.…”

Ligand Recognition Mechanism of Thiamine Pyrophosphate Riboswitch Aptamer

“…The thiamine pyrophosphate (TPP) riboswitch, which is the most widespread riboswitch among those currently known, is a representative member of type II riboswitches. Although it was one of the first riboswitches discovered and has been extensively studied for the last decade, the question of how TPP binding is coupled to the structural transition of the TPP riboswitch remains unanswered. Recent single‐molecule studies have provided valuable but controversial results.…”

Structure and Thermodynamics of Drug-RNA Aptamer Interactions

“…The similarity in energetics of binding is eclipsed by the specificity of each binding pocket created by differences in sequence and geometry of the binding pocket (Table 2). An interesting trend is observed in the investigation of small molecule binding aptamers, binding involves two stages [84,105,100]. The first stage is an interaction independent of the target and the second stage is target dependent.…”

“…TPP binds to the riboswitch between two parallel stems [99]. Investigation into the folding on this riboswitch has shown a two-step binding process involving first ligand independent secondary structure formation followed by ligand dependent tertiary structure formation [100].…”