All known guanine-sensing riboswitches regulate gene expression by specifically binding to guanine (G) or related analogs with high affinity to switch off transcription. The aptamers of this class of riboswitches are characterized by three helices (P1-P3), surrounding a central core of phylogenetically conserved nucleotides and a long-range loop-loop interaction. To gain more insight into the switching mechanism, we present here a comparison between the solution-state structures of the G-free and G-bound forms of the guanine aptamer from the xpt-pbuX operon of Bacillus subtilis, as derived from NMR chemical shifts and magnetic-field-induced residual dipolar couplings. The high-resolution NMR analysis shows the G-free aptamer is highly structured with parallel P2 and P3 helices and the long-range loop-loop interaction already present, implying that the structure is largely preformed to bind the ligand. Structural changes upon guanine binding are found to be localized to the central core. In the free state, the G-quadruple interaction and two base pairs of the P1 stem flanking the central core appear to be largely disordered. The ligand thus binds via a combined predetermined-induced fit mechanism, involving a previously unstructured five-residue loop of the J2-3 junction that folds over the ligand. These limited additional interactions within a preorganized setting possibly explain how the aptamer rapidly responds to ligand binding, which is necessary to switch the structural state of the expression platform within a narrow time frame before the RNA polymerase escapes the 59-UTR.

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL.

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The replication of Hepatitis B virus is initiated by binding of its reverse transcriptase to the apical stem loop and primer loop of epsilon. Here, we present the 1 H/ 13 C/ 15 N NMR assignments of the bases and sugars of the 29 residues apical stem loop of Duck HBV epsilon.Keywords HBV Á RNA Á Duck Á Apical loop Biological contextThe Hepatitis B virus is the most common cause of liver infection in the world, with 300 million people worldwide estimated to be chronically infected. No efficient elimination of HBV in effected patients exists as yet. HBV is a member of the Hepadnaviridae family consisting of the hepatotropic DNA viruses, which also includes related animal viruses such as duck HBV and heron HBV. The genome of HBV is a small (3.2 kb), relaxed circular, partially double stranded DNA genome which replicates by reverse transcription of an RNA intermediate, the pregenomic RNA (Beck and Nassal 2007;Girard et al. 2007). The pregenomic RNA is transported into the cytoplasm and encapsidated into immature core particles together with HBV reverse transcriptase (RT). Binding of the viral RT to the encapsidation signal, epsilon, e, a conserved 60nt bulged RNA located at the 5 0 end of the RNA pregenome, triggers encapsidation. Subsequently, a 4nt DNA primer is synthesized using the e-primer loop as template. The resulting complex translocates to a 3 0 -proximal e RNA element of the pgRNA, where full-length DNA synthesis is started using the 4-nt DNA as primer (Flodell et al. 2006;Girard et al. 2007;Petzold et al. 2007). Although the structural basis and sequence requirements for RT-e binding and priming are emerging, several questions remain and a full understanding of the molecular basis for the specific interactions between P and e awaits high-resolution structural and thermodynamic data. The importance of high-resolution structural data is underlined by the NMR studies of the human HBV e apical stem-loop, which showed that its conserved apical loop folds into a pseudotriloop, whereas secondary structure programs predicted a hexaloop (Flodell et al. 2002(Flodell et al. , 2006. A functional in-vitro RT-e replication system exists for Duck, but not for human HBV. The Duck RT-e interaction is therefore best understood, although many conclusions can be extrapolated to Human HBV thanks to the close similarity of the two HBVs (Beck and Nassal 2007). We therefore study the human and Duck e-RNAs in parallel. Here we present the NMR assignment of the Duck HBV e apical stem-loop. Methods and experimentsThe RNA sequence was prepared as previously described (Girard et al. 2007). NMR samples were prepared in buffer (10 mM Na-Phosphate, pH 6.7, 0.1 mM EDTA) giving a 1.2 mM non-labelled sample, a 1.0 mM 13 Assignments and data depositionThe assignments have been deposited in the BMRB database under number 15656 with assignment tables at three different temperatures, 5°C, 15°C and 25°C. Initial assignment was carried out according to standard methods (Flinders and Dieckmann 2006;Wijmenga and van Buuren 1998). Spect...

Here we report the nearly complete base assignments and partial sugar assignments of the 35-residue terminator hairpin of the Bacillus subtilisxpt-pbuX-mRNA guanine sensing riboswitch.