G-register exchange dynamics in guanine quadruplexes

Abstract

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially benefitting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantifies conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3 ± 1.6 J mol−1 K−1, with melting temperature increases up to 7.3 ± 1.6°C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon.

Length heterogeneity at conserved sequence block 2 in human mitochondrial DNA acts as a rheostat for RNA polymerase POLRMT activity

“…From our analysis of the human mtDNA sequences, there is clear evidence that CSB 2 varies in the number of residues in both the first and second G-runs, although >84% of the changes are in the second G-run of a G6AG n sequence, where N = 2–11 (Figure 2 ). As the total number of guanine residues was increased, there was a general trend towards increased transcription termination measured in vitro (Figure 2 ), as one would predict from an increase in quadruplex stability as more tetrads form ( 52 )). However, regardless of the variant investigated in vitro , the 3′ ends of the terminated transcripts were located at similar downstream sequences (Figure 5 ).…”

“…The quantified data and statistical variation are shown in Supplementary Figure S6. There was a general trend towards increased TP levels as the as the total length of the G-tract was increased, as one would predict from an increase in quadruplex stability as more guanine tetrads can form ( 52 ). In most cases only TP1 and TP2 bands were visible above background (Supplementary Figure S6B–D).…”

“…In addition to modulating quadruplex stability, CSB 2 length heterogeneity may result in increased heterogeneity in quadruplex structure ( 52 ). Longer sequences may also allow quadruplexes to seed in different registers; for example, there is evidence for an increased number of distinct transcription products upstream of T 284 for the G12–G18 continuous sequences in Figure 5C .…”

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

“…With another long repeat sequence, the double G-to- I substituted TAG 3 (TTAG 3 ) 7 TT, it was found that one permutation of -substitutions at the 5′-end produced a minor increase in the thermodynamic stability relative to the wild type, while four other double mutations, however, decreased it, some of them significantly [ 331 ]. G-to-I substitutions were also used with GQs of A(htel-21) and A(htel-21)T [ 332 ], the VEGFA G 3 AG 3 TTG 4 TG 3 , the c-myc Pu18 AG 3 TG 4 AG 3 TG 4 , and the PIM1 G 3 CG 4 CG 5 CG 4 sequences [ 333 ]. Guanine in the central TGT loop of the TBA GQ was replaced by adenine or hypoxanthine to study the role of G8 in the GQ stabilization.…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…It is interesting that "excess" of guanosines in some G tracts can cause their sliding relative to each other during formation of quadruplex structures. It was shown that this process stabilized the folded state; the T m value of G4 increased significantly due to favorable entropic changes [136].…”