The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

Abstract

The N protein from bacteriophage is a key regulator of transcription antitermination. It specifically recognizes a nascent mRNA stem loop termed boxB, enabling RNA polymerase to read through downstream terminators processively. The stacking interaction between Trp-18 of WT N protein and A7 of boxB RNA is crucial for efficient antitermination. Here, we report on the direct probing of the dynamics for this interfacial binding and the correlation of the dynamics with biological functions. Specifically, we examined the influence of structural changes in four peptides on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in different positions), through mutations of critical residues of N peptide (residues 1-22). We then compare their in vivo (Escherichia coli) transcription antitermination activities with the dynamics. The results demonstrate that the RNA-peptide complexes adopt essentially two dynamical conformations with the time scale for interfacial interaction in the two structures being vastly different, 1 ps for the stacked structure and nanosecond for the unstacked one; only the weighted average of the two is detected in NMR by nuclear Overhauser effect experiments. Strikingly, the amplitude of the observed ultrafast dynamics depends on the identity of the amino acid residues that are one helical turn away from Trp-18 in the peptides and is correlated with the level of biological function of their respective full-length proteins.M acromolecular assemblies play critical roles in biological processes, such as expression (1), recognition (2, 3), and catalysis (4, 5). In these assemblies, the interactions are selective in forming unique structures with dynamics governing the functions. One such complex is that of RNA with proteins. It involves the bacteriophage N protein, which binds a cis-acting stem-loop RNA structure (termed boxB) in nascent mRNA coded in the phage genome, and regulates transcription elongation and termination (6-8). With other host protein factors, the complex enables RNA polymerase to read through intrinsic and Rho-dependent terminators in a processive manner (9).NMR structural studies (10-12) demonstrated that the amino terminal of the N protein (N peptide), which features an arginine-rich motif, binds to the boxB RNA hairpin as a bent ␣-helix, and this process enforces the purine-rich boxB RNA pentaloop to adopt a canonical GNRA fold (13, 14) with the fourth purine residue extruded (Fig. 1). One unique interaction featured in this RNA-peptide complex is that of the side chain of the tryptophan residue (Trp-18), which directly stacks on adenine 7, extending the RNA -stack by one residue (Fig. 1). Different peptide ligands can target boxB RNA with strong equilibrium affinities (nanomolar range for dissociation constant) similar to the WT (15), as we have shown by in vitro protein selection via mRNA display (16,17). However, examination of the energetics and structures by NMR, CD, and steady-state fluorescence on some of the selected 14͞15 mutants (Fig. 1) reveal that they have quit...

4d Ultrafast Electron Diffraction, Crystallography, and Microscopy

“…Similarly, for RNA recognition of proteins (Figure 3), the dynamics of the complex was found to be interfacial and is controlled by a single residue, the interface between tryptophan of polypeptides and bases of RNA (13,14). The recognition is critically dependent on conformational structures, stacked (active) or unstacked (inactive).…”

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

“…1A,D,E). The stacking of Tryptophan 18 of the N-peptide on adenine 7 of the boxB pentaloop (Trp18/A7 stacking) is known to be a critical factor that affects N-peptide's biological function of transcription anti-termination (Austin et al 2003;Xia et al 2003aXia et al ,b, 2005.…”

“…However, these mutations significantly impact the functional capacity of N protein variants. For example, mutating the wild-type sequence of K 14 Q 15 to E 14 R 15 completely abolished anti-termination activity when measured in vivo (Xia et al 2003a). Based on ultrafast time-resolved spectroscopic probing (Xia et al 2003a(Xia et al , 2005, a dynamic two-state model has been proposed, which stipulates that the N-peptide/boxB complex exists in dynamic equilibrium between stacked and unstacked states (Fig.…”

“…It has been proposed that changes in the percentage of the stacked state account for the functional variations of the mutations (Xia et al 2005). These studies suggest a dynamic recognition mode at the N-peptide/boxB protein-RNA interface, where a single stacking interaction (i.e., Trp18/A7) seems to be critical for the entire anti-termination machinery (Xia et al 2003a(Xia et al ,b, 2005.…”

“…The existence of at least two discrete states in the N-peptide/boxB complex is inferred from ultrafast timeresolved spectroscopic measurements that monitor femtosecond to picosecond fluorescence decay of 2-aminopurines (2AP) incorporated in the boxB stem-loop (Xia et al 2003a(Xia et al , 2005. The nature of the bound peptide structure, particularly in the unstacked state, is not clear because fluorescence signals came from the RNA portion of the complex.…”