RESUMO
Here we report the nearly complete base assignments and partial sugar assignments of the 35-residue terminator hairpin of the Bacillus subtilis xpt-pbuX-mRNA guanine sensing riboswitch.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana Transportadoras/genética , RNA Bacteriano/química , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico , Bacillus subtilis , Isótopos de Carbono/química , Guanina/metabolismo , Hidrogênio/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genéticaRESUMO
S-Adenosyl-L-methionine (SAM) is the preferred cofactor for biological methyl group transfers to various substrates such as nucleic acids, proteins, and lipids. Here we present stereospecific (>95% of the desired enantiomer) and high-yield preparation of four fluorescent and biologically active SAM analogs and demonstrate their usefulness in binding studies. Using a fluorescence titration experiment, we obtained a K(d) of 0.38 microM for the S-2,6-diaminopurinylmethionine-SAM-III riboswitch complex.
Assuntos
Corantes Fluorescentes/química , S-Adenosilmetionina/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/química , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
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 5'-UTR.
Assuntos
Guanina , RNA Bacteriano/química , Sequências Reguladoras de Ácido Ribonucleico , Adenina/química , Adenina/metabolismo , Aptâmeros de Nucleotídeos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Pareamento de Bases , Sequência de Bases , Cátions Bivalentes/farmacologia , Guanina/química , Guanina/metabolismo , Ligantes , Magnésio/farmacologia , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Óperon , RNA Bacteriano/efeitos dos fármacos , RNA Bacteriano/genéticaRESUMO
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 degrees 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.