G-Quadruplexes: More Than Just a Kink in Microbial Genomes

Abstract

G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures formed by guanine-rich DNA and RNA sequences. In this review we aim to provide an overview of the biological roles of G4s in microbial genomes with emphasis on recent discoveries. G4s are enriched and conserved in the regulatory regions of microbes, including bacteria, fungi, and viruses. Importantly, G4s in hepatitis B virus (HBV) and hepatitis C virus (HCV) genomes modulate genes crucial for virus replication. Recent studies on Epstein-Barr virus (EBV) shed light on the role of G4s within the microbial transcripts as cis-acting regulatory signals that modulate translation and facilitate immune evasion. Furthermore, G4s in microbial genomes have been linked to radioresistance, antigenic variation, recombination, and latency. G4s in microbial genomes represent novel therapeutic targets for antimicrobial therapy.

Keywords: G-quadruplexes; G4s; bacteria; biological functions; microbes; viruses.

Figure 1

Structure of a G-Quadruplex (G4). (A) The nucleotide sequence of a G4 motif with varicoloured G-triplets separated by loop sequences (L₁, L₂, L3). (B) A 2D representation of a typical G4 fold showing the three planar quartets. The spheres at the vertices of the quartets represent one G from each of the four G-triplets. The black sphere at the centre denotes the central metal cation (Na⁺, K⁺) needed to stabilize the G4 structure. (C) A top view of a planar G-quartet showing the Hoogsteen bonds (dashed lines), the atoms thereof, and a cation in the central cavity. Figures are not drawn to scale.

Figure 2

Molecular Mechanisms Affected by G-Quadruplexes (G4s) in Microorganisms. The functional roles of G4s identified in microbes are included in this graph. The segments in the outermost circle (blue) denote the molecular biological processes regulated by G4s. The segments of the intermediate circle (orange) indicate the types of microbe affected for each biological process (see the legend beside the graph). The specific microorganism under each type is denoted either by an abbreviation (for viruses) or a three-letter notation (for bacteria and parasites) in the vertical bars in the innermost circle (see the legend beside the graph). The vertical bars in the innermost circle are colour-coded for bacteria (green), viruses (red), and parasites (black). The curved lines connect a given microbe with multiple quadruplex-regulated biological processes. Red curved lines connect viruses, and blue curved lines connect parasites.

Figure 3

The Link between G-Quadruplexes (G4s) and Microbial Evolution. The conservation or elimination of G4s from specific genomic locations within a species or across related species of microbes implicates these nucleic acid secondary structures in microbial evolution. Besides, G4s directly participate in mechanisms that generate diversity in the microbial populations, such as antigenic variation and recombination. E. coli, Escherichia coli; S. cerevisiae, Saccharomyces cerevisiae.

Figure 4

Roles of G-Quadruplexes (G4s) in Human Herpesviruses (HHVs). (A) Quadruplexes (red) present in the promoter regions of the HHVs negatively regulate gene expression. Addition of G4-binding ligand (blue) further augments the inhibition of gene expression. (B) HHV genomes exist as concatemers during replication. A quadruplex formed at the pac-1 signal (packaging signal) acts as a scaffold for the protein machinery that cleaves and encapsidates unit-length genomes. The sites of cleavage proximal to the quadruplex-forming pac-1 signals are shown in orange. (C) Binding of ligands (blue objects) to G4s (red triangular kinks) present near the origin of bidirectional episomal replication (red bulge with two-headed arrow) in the terminal repeat region (red portion) of Kaposi’s sarcoma-associated herpesvirus (KSHV) episome, prevents progression of DNA polymerase (green ovals). The slippage of the polymerase leads to replicative stress that activates dormant origins (grey bulges on either sides). The net effect of the ligand binding to quadruplexes is a reduction in the number of KSHV episomal copies. (D) Quadruplex (red) formed in the Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1) mRNA causes stalling of the ribosome-nascent chain complex (green and blue), resulting in inhibition of translation. EBV is thus able to maintain the levels of EBNA-1 below the detection threshold, evading immune response during latency. The binding of nucleolin (orange) to the quadruplex further reduces EBNA-1 protein levels. (E) Ligands (blue objects) bind to G4s (red) in the herpes simplex virus-1 (HSV-1) genome and stabilize them, causing polymerase (green ovals) stalling on the leading and lagging strands, inhibiting HSV-1 DNA replication. Arrows indicate direction of replication. Figures A through E are not drawn to scale.

Figure 5

Phenotypic Effects of the Modulation of Transcription by G-Quadruplexes (G4s). G4-mediated control of microbial transcription appears to be a common theme leading to tangible differences in the phenotype of microbes. G4s in microbial genomes may regulate microbial transcription either negatively (pink circles) or positively (green circle). D. radiodurans, Deinococcus radiodurans; P. denitrificans, Paracoccus denitrificans; EBOV, Ebola virus.