Occurrence of a quadruplex motif in a unique insert within exon C of the bovine estrogen receptor alpha gene (ESR1).

The 5' end of exon C of the bovine estrogen receptor alpha gene (bov-ESR1) includes a unique G-rich insert, not found in other closely related mammalian genes, which lies close to both a double E-box transcription factor binding site and the site of a single nucleotide (G/A) polymorphism. Biophysical studies, using CD and UV absorbance measurements, show that this 22 base insert leads to the formation of a family of stable G-quadruplex structures which are unaffected by the G/A polymorphism. Multiplex PCR shows that the region including the G-quadruplex is transcribed into RNA, and studies with a synthetic RNA transcript sequence demonstrated formation of a highly stable parallel-folded quadruplex structure. Luciferase reporter constructs demonstrate that the G-rich sequence reduces rates of translation when present in the 5'-UTR of mRNA transcripts. Mutations (GGG to AAA) that destabilize the quadruplex lead to a 15-fold enhancement of translational efficiency, suggesting that a possible biological role of the insert in exon C of the bov-ESR1 is to regulate translation of this exon.

Repression of translation of human estrogen receptor alpha by G-quadruplex formation.

Tissue-specific expression of the human estrogen receptor alpha gene (ESR1) is achieved through multiple promoter sequences resulting in various mRNA transcripts encoding a common protein but differing in their 5'-untranslated region (5'-UTR). Many cancers are estrogen-sensitive with neoplastic growth stimulated through the estrogen receptor, a transcription factor that regulates developmental genes. We demonstrate that the human ESR1 gene is rich in potential quadruplex-forming sequences with 3 of 20 identified within exonic regions. In particular, we show using CD, UV, and NMR spectroscopy that a stable DNA G-quadruplex motif is formed within the exon C gene sequence. This motif, which PCR shows is transcribed in normal and neoplastic endometrium and in MCF-7 cells, forms a stable RNA quadruplex demonstrable by CD and UV analysis. Cloning the exon C G-quadruplex sequence upstream of a luciferase reporter gene caused a 6-fold reduction of enzymatic activity compared to a mutant sequence. We conclude that the exon C G-quadruplex motif is present in the 5'-UTR of the mRNA transcript, where it modulates the efficiency of translation.

Folding of single-stranded DNA quadruplexes containing an autonomously stable mini-hairpin loop.

The single-stranded DNA quadruplex motif TG(3)-L(1)-G(3)-L(2)-G(3)-L(3)-G(3)T (where L(1), L(2) and L(3) are the three loop sequences) was used as a template for probing the effects of the loop sequences on stability and folding topology. An autonomously stable mini-hairpin sequence (ACGTAGT) was inserted into the central loop (L(2)) of different sequences with intrinsic propensities to form either parallel or anti-parallel structures. Single nucleotides (T) at positions L(1) and L(3) strongly favour the formation of a parallel structure with the L(2) hairpin insert affecting stability in the same way as a T(7) loop. However, in the context of an anti-parallel quadruplex with T(3) loops in positions L(1) and L(3), the mini-hairpin in the central loop forms a stable structure which enhances the T(m) of the quadruplex by approximately 10 degrees C when compared with the T(7) insert. The CD and UV melting data show that base pairing interactions within the ACGTAGT hairpin loop sequence, when accommodated as a diagonal loop in an anti-parallel structure, can enhance stability and lead to novel quadruplex structures, adding complexity to the folding landscape and expanding the potential repertoire of sequences that are able to regulate gene expression in vivo.

Folding topology of a bimolecular DNA quadruplex containing a stable mini-hairpin motif within the diagonal loop.

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G(3)ACGTAGTG(3))(2) containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G(3)T(4)G(3))(2) dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T(4)-loop sequence. A truncated version of the bimolecular quadruplex d(G(2)ACGTAGTG(2))(2), with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.

The highly repetitive region of the Helicobacter pylori CagY protein comprises tandem arrays of an alpha-helical repeat module.

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagY(rpt2)) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagY(rpt2) to comprise two highly thermostable and acid-stable alpha-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagY(rpt2) structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagY(rpt2) assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagY(rpt2) region.

Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria.

Hairpins are known to play specific roles in DNA- and RNA--protein recognition. Various disease states are thought to originate from the ill-timed formation of a hairpin loop during transcription, particularly in the context of triplet repeats which are associated with myotonic dystrophy, fragile X syndrome and other genetic disorders. An understanding of nucleic acid folding mechanisms requires a detailed appreciation of the timescales of these local folding events, a characterisation of the conformational equilibria that exist in solution and the influence of point mutations on the relative stabilities of the different species. We investigate using NMR and CD spectroscopy the structure and dynamics of a DNA hairpin containing a highly stabilising cGNAg loop. The single-stranded 13-mer 5'-d(GCTACGNAGTCGC) with N = T folds to form a hairpin structure which accommodates a C-T mis-matched base pair within the double-stranded stem region. The hairpin is in equilibrium with a double-stranded duplex form with the mixture of two interconverting conformations in slow exchange on the NMR timescale (1-2 s(-1) at 308 K). We are able to characterise the dynamics of the interconversion process by NMR magnetisation transfer and by CD stopped-flow kinetic experiments. The latter shows that the hairpin folds too rapidly to detect by this method (>500 s(-1)) and forms in a "kinetic overshoot" followed by a much slower equilibration to a mixture of conformations ( approximately 0.13 s(-1) at 298 K). A point mutation that converts the GTA to a GAA loop sequence destabilises the intermolecular duplex structure and enables us to unambiguously assign the various dynamic processes that are taking place.