Abstract
Nun protein from coliphage HK022 binds to phage boxB RNA and functions, in contrast to phage lambda N protein, as a transcriptional terminator. The basic Nun-(10-44) peptide contains the boxB RNA binding arginine rich motif, ARM. The peptide binds boxB RNA and competes with the phage lambda ARM peptide N-(1-36) as indicated by nuclear magnetic resonance (NMR) spectroscopy titrations. In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does in complex with N peptides containing the ARM, and we could show that Nun-(20-44) forms a bent alpha-helix upon binding to the boxB RNA. The structure of the boxB RNA-bound Nun-(20-44) was determined on the basis of 191 intra- and 30 intermolecular distance restraints. Ser-24 is anchored to the lower RNA stem, and stacking of Tyr-39 and A7 is clearly experimentally indicated. Arg-28 shows numerous contacts to the RNA stem. Leu-22, Ile-30, Trp-33, Ile-37, and Leu-41 form a hydrophobic surface, which could be a recognition site for additional host factors such as NusG. Such a hydrophobic surface area is not present in N-(1-36) bound to boxB RNA.
Highlights
Bacteriophage N protein plays an essential role in transcriptional antitermination in the two-phage early operons that are critical for phage development
In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20 – 44) exhibits the same pattern of resonances as it does in complex with N peptides containing the arginine-rich motif (ARM), and we could show that Nun-(20 – 44) forms a bent ␣-helix upon binding to the boxB RNA
The structures of phage N ARM peptide-boxB RNA complexes and of a phage P22 ARM N peptide-boxB RNA complex have been solved by nuclear magnetic resonance (NMR) (9 –12)
Summary
The inhibition of termination at intrinsic and -dependent terminators by N protein depends on recognition of an RNA element called nut (N utilization) on the nascent phage transcript and on four Escherichia coli host factors (NusA, NusB, NusG, and ribosomal protein NusE). Together they form a ribonucleoprotein complex that converts the RNA polymerase into a termination-resistant. It has been proposed that Nun arrests transcription by anchoring RNA polymerase to DNA (8) Both Nun and N proteins belong to the family of arginine-rich motif (ARM) binding proteins. For both these phage peptides, a very similar mode of binding has been observed, with the peptides bound in the major groove of boxB RNA, which adopts a typical hairpin conformation closed by an apical tetraloop
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