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

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 be located within an operon, between the promoter and the terminators (2). Two components have been identified within nut: 8-base boxA, 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 lambda'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 lambda. These observations extend previous mutational knowledge of nut (7) and are consistant with the hypothesis that 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.