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Title: Structure and interaction of telomeric and telomerase RNA G-quadruplexes.
Authors: Martadinata, Herry.
Keywords: DRNTU::Science::Biological sciences::Biophysics
Issue Date: 2013
Abstract: G-rich oligonucleotides are able to form four-stranded nucleic acid structures called G-quadruplexes in cationic solution. These structures are composed of multiple tetrad planes, in which each tetrad plane consists of four guanines that are Hoogsteen base-paired to one another. Bioinformatics studies across the human genome have found about 376,000 putative G-quadruplex forming sequences. In this work we elucidate the structures and interactions of G-quadruplexes formed by human telomeric RNA (TERRA) and human telomerase RNA sequences. TERRAs are transcript products of the telomere. The sequence of TERRA is composed of the sequences from the sub-telomere region and multiple repeats of UUAGGG. TERRAs were shown to perform various regulatory functions in telomere biology. In our study we show that repeats of UUAGGG are able to form G-quadruplex structures in potassium solution. In this thesis, we present the high resolution NMR structure of G-quadruplex formed by a two-repeat 12-nt human TERRA sequence, r(UAGGGUUAGGGU) in potassium solution. The 12-nt human TERRA sequence forms a dimeric parallel-stranded three-layer G-quadruplex in potassium solution. Mixtures of C2’-endo and C3’-endo sugar puckering were observed for the sugar bases. We also solved the high resolution structure of G-quadruplex formed by 10-nt human TERRA sequence, r(GGGUUAGGGU) in potassium solution. The 10-nt human TERRA sequence forms a six-layer tetrameric parallel-stranded G-quadruplex in potassium solution. The absence of UA bases in the 5’ terminal caused the two blocks of the dimeric G-quadruplexes to stack at their 5’ end. The Adenines in the UUA loops were also re-arranged to be co-planar with the 5’-end G-tetrad forming an A:(G:G:G:G):A hexad stacking interface. A Higher order arrangement of G-quadruplexes in long TERRA context was proposed based on this stacking arrangement. TERRAs are transcribed with length ranging from 100-9000 nt. Multiple G-quadruplex structures can be formed along the long human TERRA sequences. Our study showed the “beads-on-a-string” arrangement of G-quadruplexes in long human TERRA. Each bead is composed of either four-repeat or eight-repeat that presumably form single block and two-block stacked three-layered parallel-stranded G-quadruplexes respectively. MD simulations of four possible stacking interfaces, i.e. 5’-5’, 5’-3’, 3’-5’ and 3’-3’ demonstrated feasibility of all the arrangements except for the 5’-3’. MD simulation of the 5’-5’ stacking arrangement revealed continuous stacking of loops bases across the two stacked blocks. This loop arrangement might contribute to the overall stability of the structures. TERRAs were reported to co-localize to telomeric DNA during interphase and metaphase. A TERRA-telomere DNA hybrid G-quadruplex was proposed as one of the possible association modes. Our studies show that the dissociation constant (Kd) of DNA-RNA hybrid G-quadruplexes is virtually independent on the sequences of DNA but instead depends on the number of G-repeats of the DNA and RNA sequences. The sugar-ring chemistry (deoxyribose vs. ribose) was found to affect the Kd of inter-molecular G-quadruplex (DNA-DNA vs. DNA-RNA vs. RNA-RNA intermolecular G-quadruplex). Protein hnRNP A1/UP1 were reported to interact with telomeric DNA and TERRA in vivo. hnRNPA A1/UP1 were previously shown to unfold DNA G-quadruplexes from various sequences in vitro. Here we present that protein UP1 binds and unfolds both telomeric DNA and RNA G-quadruplexes. Telomeric DNA G-quadruplex was found to be unfolded faster than telomeric RNA G-quadruplex. The 5’-end G-rich sequences of human telomerase RNA was reported to be capable of forming G-quadruplexes in vitro. The formation of this structure was thought to be important in telomerase biology. In this work we show the formation of G-quadruplex by the first 18-bases of the 5’ human telomerase RNA sequence in potassium solution. The G-quadruplex formed is an intra-molecular three-layer parallel-stranded G-quadruplex. The G-quadruplex formed has a higher thermal stability than the G-quadruplex formed by human telomeric RNA sequences. The stacked G-quadruplex dimer conformation was observed at a high potassium or high RNA concentration. Our results contribute to the understanding of structure, stability and interaction of telomeric and telomerase RNA G-quadruplexes. This might aid in further explicating their biological roles and regulatory mechanisms. Our results are valuable in designing small-molecule targeting these molecules or their interaction partners.
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