NMR studies on structures of E. coli RBP signal sequences and RNA recognition by Drosophila Sex-lethal protein

Abstract

(Part Ⅰ) It was recently reported that the α-helical content of the signal peptide of Escherichia coli ribose binding protein (RBP), as determined by circular dichroism (CD) and 2D NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide [G. Yi, B.-S. Choi, and H. Kim (1994) Biophys. J. 66, 1604-1611]. In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were determined also using CD and 2D $^1H$ NMR spectroscopy. CD results showed that both of these revertant signal peptides have an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher. On the other hand, the helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same. This discrepancy may be due to the stability difference between identical helical stretches in wild-type and revertant peptides. A good correlation between the helical content of these four RBP signal peptides in TFE/water as studied by CD and their in vivo translocation activities was observed. It appears, therefore, that both the proper length of helix and the stability are of functional significance. (Part Ⅱ) The interactions of the second RNA-binding domain of Drosophila melanogaster Sex-lethal protein (Sxl RBD2) with oligoribonucleotides, GUUUUUUUU ($GU_8$) and CUAGUG, the sequences present around an alternative 3`` splicing site of the tra pre-mRNA ($GU_8$CUAGUG), were studied by using heteronuclear two-dimensional NMR techniques. The $^1H$ and $^15N$ chemical shifts of the backbone amide resonances upon titration of the Sxl RBD2 with each of these RNAs were recorded. It was found that the Sxl RBD2 can bind not only to the polyuridine tract, $GU_8$, but also to the downstream 3``-splice site sequence, CUAGUG, with similar binding affinities. In...