Untitled

Abstract

We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.

Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element

“…The equine infectious anemia virus RRE and the human endogenous retrovirus type K RcRE also may bind their respective regulatory proteins in two distinct regions (15,38). Use of Rev peptides and an oligomer derived from HIV stem-loop IIB have indicated considerable flexibility of the RRE (42). Purified Rem protein has been difficult to produce, but preliminary data using filter-binding assays indicate that Rem SP binds with comparable affinities to both the full-length and RmRE-(1-348).…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…Alternatively, the affinity purification tag can be separated from the protein of interest. In the case of small peptides, removal of the affinity purification tag was often achieved using cyanogen bromide that specifically hydrolyzes peptide bonds C-terminal of methionines [7,9,15,16,19,33,42,44]. In the case of protein domains that often contain internal methionines, other methods for cleavage are generally used and involve specific proteases.…”

“…13A). Fortunately, the part of highest interest, namely the RNA at the binding interface, often experiences large chemical shift changes, leading to a larger dispersion of the correlations and likewise cytosine amino protons H41/H42 were correlated to H5 protons using H5-N4, H41-N4 and H42-N4 correlations [16].…”

“…The HNN-COSY has been used for the structure determination of protein-RNA complexes ranging from to 10 to 28 kDa [9,16,22,36]. A detailed protocol for the HNN-COSY TROSY was recently published [217].…”

“…Indeed, 19 out of 22 structures of viral protein-RNA complexes were solved by NMR [4, 6, 8, 10-13, 16, 19, 23, 31, 41-45]. Three types of viral protein-RNA complexes were studied: the complexes between the viral protein Rev and its RNA target, the RRE, involved in RNA export [6,16,19,41,42,44] ; the complexes between the viral protein Tat (trans-activator) protein and its RNA target, the TAR (Trans-Activator Response element), involved in transcription regulation [8,11,23,31,43] ; the complexes between the viral protein NC and its RNA target, the Ψ-site, involved in RNA packaging [4,10,12,13,45].…”