Characterization of the dual start motif of a class II holin gene

Abstract

Holins are small membrane proteins, which, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (3 for class I and 2 for class II). The prototype class I holin gene, S[], has a dual start motif and encodes not only the effector holin S[]105 but also an inhibitor S[]107 with a Met-Lys...extension at the N-terminus. The prototype class II holin gene of phage 21, S²¹, begins with the motif Met-Lys-Ser-Met...; a potential RNA secondary structure overlaps the Shine-Dalgarno sequence. Here, I demonstrate that (1) two protein products are elaborated from S²¹, S²¹71, and S²¹68; (2) the shorter product is required for lysis; (3) the longer product, S²¹71, inhibits S²¹ function; and (4) the Lys₂ residue is important for the inhibitor function. Moreover, it is shown that the RNA stem-loop structure is involved in the down-regulation of S²¹71 synthesis and that in S²¹ different segments of the single consensus Shine-Dalgarno sequence serve the two translational starts. It is concluded that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes but have a fundamentally different molecular basis.