Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of lambda

Abstract

Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB(left) complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and lambda N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by lambda N. Point mutations can dramatically alter boxB specificity between P22 and lambda N. A boxB specific for P22 N can be mutated to lambda N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

FIG. 1.

Comparison of λ and P22 nut site RNAs and N protein RNA-binding domains. (A) The secondary structures of boxBs found in bacteriophages λ and P22 are represented from the beginning of the boxB stem. LL, λ boxB_left; LR, λ boxB_right; PL, P22 boxB_left; PR, P22 boxB_right. Lightface nucleotides are found in either λ or P22 boxB sequences, while boldface nucleotides are found only in λ boxBs or only in P22 boxBs. The C:C pair adjacent to the loop of P22 boxB_right is presumed to be stacked into the helix, perhaps as a hydrogen-bonded, nonstandard base pair. (B) Sequences of λ and P22 nut sites, where boxA is bold and boxB is underlined. λL, λ nut_left; λR, λ nut_right; P22L, P22 nut_left; P22R, P22 nut_right. In this study, all boxBs were placed in the context of λ nut_left to limit the effect of differences between boxAs and the boxA-boxB interval. (C) The sequences of the RNA-binding domains of λ N and the P22 N fusion plasmid used in this study are shown aligned from their amino termini to the fusion point with the common remainder of λ N. The italicized asparagine at the far right replaces a wild-type lysine in λ N to allow a restriction enzyme site in the N-supplying plasmids. Amino acids shown in bold are conserved between λ and P22 N.

FIG. 2.

Structures and schematics of λ and P22 boxBs viewed from the minor groove. (A) The NMR structure of λ N-boxB_right (Protein Data Bank accession no. 1QFQ [39]) (top), and that of P22 N- boxB_left (Protein Data Bank accession no. 1A4T [5]) (bottom) are displayed in truncated form. The peptide is rendered in light-gray van der Waal radius spheres and the loop regions of boxBs are rendered as black lines. (B and C) Base identities and numbering superimposed on structural schematics, showing the λ 4-out and P22 3-out GNRA-like pentaloop conformations. Both have a GAAA tetralooplike structure (9). λ boxB_right is above, and P22 boxB_left is below. Open boxes represent bases; filled black boxes represent the contacting hydrophobic surface of the N protein.

FIG. 3.

Specificities of mutant boxBs. Representative boxB reporters in this study are plotted by their percentages of the activities with P22 N (abscissa) and λ N (ordinate) relative to those with P22 boxB_left and λ boxB_right, respectively (Table 6). All clones are as in Tables 1 to 5. (A) boxBs related by C-to-A changes at position 3 are shown connected (D22 to λ boxB_left [LL], D18 to λ boxB_right [LR], P22 boxB_left [PL] to D2, L4 to D7, D5 to D6). boxBs with C₃ are shown as filled circles, and boxBs with A₃ are shown as open circles. (B) boxBs related by P22 boxB_left-to-λ boxB stem changes are shown connected (D4 to LL, D2 to LR, PL to D18, L2 to D21, D3 to D20), where boxBs with P22 boxB_left stems are shown as filled circles and boxBs with λ boxB stems are shown as open circles. (C) boxBs related by U:A-to-A:U changes adjacent to the loop are shown connected (LR to D19, D3 to D7, D2 to D6, D1 to L4, PL to D5), where boxBs with U:A pairs are shown as filled circles and boxBs with A:U pairs are shown as open circles. (D) Selected boxBs related by single-nucleotide changes are shown connected (LL to D22, LR to D18, LR to D20, LR to D21, PL to D1, PL to D2, PL to L2, PL to L15, PL to D16, L4 to D7, D5 to D6, D6 to D7). Wild-type boxBs are represented by filled circles, and single-substitution mutants are shown as open circles. In the case of P22 boxB_left, single-insertion mutants are shown as open squares. Four mutant boxBs related by single substitutions are shown as open triangles.