Effects of all single base substitutions in the loop of boxB on antitermination of transcription by bacteriophage λ's N protein

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Abstract
The ’N‘ antitermination proteins of lambdoid bacteriophages are essential for overcoming multiple transcription terminators located within the major early operons of these phages (1). In order for N proteins to function, a genome sequence specifying N utilization, nut, must belocated within an operon, between the promoter and the terminators (2). Two components have been identified within nut: 8-base baxA, conserved among different phages and implicated in the recognition of host NusA protein, required for N function (3); 15-base boxB, an interrupted palindrome (4), diverged in sequence among different lambdoid phages and hypothesized to be the site of recognition for different N proteins, also diverged in sequence (5). Here we apply a plasmid for testing termination and antitermination of transcription (6) to identify mutations at all positions in the 5–7 base loop of λ's boxB. Almost every base change at any position within the 5–7 base boxB loop was found to constrain antitermination of transcription by the N protein of bacteriophage λ. Theseobservations extend previous mutational knowledge of nut(7) and are consistant with the hypothesisthat the boxB loop is the direct site of recognition for N protein. Variations among the effects of different base changes suggest differential contacts between N protein and bases of the boxB loop, whether in DNA or RNA.