RNA Recognition by a Bent α-Helix Regulates Transcriptional Antitermination in Phage λ

Su, Leila; Radek, James T.; Hallenga, Klaas; Hermanto, Patrick; Chan, Ging; Labeots, Laura A.; Weiss, Michael A.

doi:10.1021/bi971408k

Cited by 65 publications

(99 citation statements)

References 56 publications

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“…The two labeling positions were chosen based on previous studies to minimize potential disruptions to the native conformation(s) of the N-peptide/boxB complex (Su et al 1997). In the NMR structure of the N-peptide/boxB complex, side chains of these two positions point away from the peptide/RNA interface ( Fig.…”

Section: Experimental Design and Biochemical Characterizationmentioning

confidence: 99%

“…Circular dichroism (CD) measurements indicated that the N-peptide is disordered and only folds when bound to boxB RNA . NMR studies (Su et al 1997;Legault et al 1998;Schärpf et al 2000) revealed that in the bacteriophage l N-peptide/boxB complex, N-peptide adopts a bent a-helical conformation, while the boxB pentaloop (G6A7A8A9A10) folds into a stable GNRA tetraloop with the fourth loop base (A9) extruded out (Fig. 1A,D,E).…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Lee²,

Zhao³

et al. 2010

RNA

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In bacteriophage l, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.

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Section: Experimental Design and Biochemical Characterizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Zhang

Lee²,

Zhao³

et al. 2010

RNA

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“…The methyl group of Ala13 lies between C7 and C8, packing against both the ribose and base H5/H6 protons of these nucleotides. This alanine is conserved among the family of N proteins, and mutagenesis of this position in the N system showed that only alanine and serine maintained wild-type binding affinity for the boxB RNA (Su et al 1997). Tyrosine-17 is one turn up the ␣-helix and lies between U9 and C10, the next two bases up the stem from the Ala13 position.…”

Section: Van Der Waals Contactsmentioning

confidence: 99%

“…The N peptide has a pronounced bend in the ␣-helix of ∼120°as a result of deviations of the backbone and angles for Arg11. This bend has been shown to be important for N binding (Tan and Frankel 1995;Su et al 1997) and antitermination in vivo (Franklin 1993;Su et al 1997), and is required for bringing Trp18 into position for stacking onto the boxB tetraloop. The N P22 peptide ␣-helix is also bent, peptide ␣-helix is almost linear.…”

Section: Overview Of 21 and P22 N Peptide-boxb Structuresmentioning

confidence: 99%

Structural mimicry in the phage [phis]21 N peptide–boxB RNA complex

Cilley

Williamson²

2003

RNA

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We determined the solution structure of a 22-amino-acid peptide from the amino-terminal domain of the bacteriophage 21 N protein in complex with its cognate 24-mer boxB RNA hairpin using heteronuclear magnetic resonance spectroscopy. The N peptide binds as an ␣-helix and interacts predominately with the major groove side of the 5 half of the boxB RNA stem-loop. This binding interface is defined by surface complementarity of polar and nonpolar interactions, and little sequence-specific recognition. The 21 boxB loop (CUAACC) has hydrogen bond and backbone torsions typical of the "U-turn" motif, as well as base stacking of the last 4 nt, and a hydrogen bonded C:C pair closing the loop. The exposed face of the 21 boxB loop, in complex with the N peptide, is strikingly similar to the GNRA tetraloop-like folds of the related and P22 bacteriophage N peptide-boxB RNA complexes. The N peptide-boxB complexes of the various phage, while individually distinct, provide similar structural features for interactions with the Escherichia coli host factors to enable antitermination.

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“…NMR structural studies (10)(11)(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).…”

mentioning

confidence: 99%

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

Xia

Becker

Wan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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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...

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RNA Recognition by a Bent α-Helix Regulates Transcriptional Antitermination in Phage λ

Cited by 65 publications

References 56 publications

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

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

Structural mimicry in the phage [phis]21 N peptide–boxB RNA complex

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

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