HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N

Abstract

Bacteriophage N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to N, the Nun protein of HK022 binds the boxBs of coinfecting transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and N antitermination complexes, the same Nun libraries were fused to the activation domain of N and screened for clones able to complement N-deficient . Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions. T he switch to delayed early gene expression in , P22, 21, and other lambdoid bacteriophages depends on N proteins assembling antitermination complexes at nut (N utilization) sites on P left and P right transcripts that transcribe past downstream terminators (1-3). N proteins bind via their arginine-rich motifs (ARMs) to boxB hairpin RNAs in nut sites of P left and P right transcripts ( Fig. 1) (4,5) and interact with host factors in the transcription elongation complex, including NusA, NusB, NusE,. N antitermination has been extensively studied, and detailed biophysical (9-11), mechanistic (12)(13)(14), and structural models are published (15)(16)(17).In competition with bacteriophage , HK022 uses its Nun protein to suppress the replication of coinfecting by premature termination of P left and P right transcripts (18-21). Similarly to N, Nun binds boxBs via its ARM in an elongation complex that also includes NusA, NusB, NusE, and NusG (6,17,20,[22][23][24][25]. Based on Nun's conserved ARM sequence, its inability to exclude P22 and 21 infections (18, 26), the affinity of Nun ARM-boxB in vitro (27,28), and the similarity of Nun's ARM-boxB NMR structural model (29) to those of N (15, 16), the recognition strategy of the HK022 Nun ARM-boxB interaction has been assumed to be very similar or identical to that of the N-boxB interaction, though few Nun ARM mutants have been ex...

Rational Design of Aptamer-Tagged tRNAs

“…By using the H072 chassis and E. coli HisRS, several cognate pairs of RNA aptamers and protein domains [ 2 , 28 , 50 , 53 , 54 , 55 ] were examined ( Figure S5 A,B). The first preliminary experiment revealed that the cognate pair of the 7SK RNA stem-loop 4 (SL4) and the hLarp7 xRRM domain [ 53 ] fused to the N -terminus of E. coli HisRS ( Figure 6 D,E) might have enhanced the His-inserting activity by the tRNA-aptamer fusion in E. coli expressing the cat(His193TAG) gene ( Figure S5 C).…”