Analysis of the spacial requirements for RNA-protein interactions within the N antitermination complex of bacteriophage lambda.

In bacteriophage lambda, formation of a transcriptional antitermination complex consisting of the lambda N protein, nut RNA transcript (boxA-boxB), host factors, and RNA polymerase is mediated by the interaction of the boxB RNA with the RNA-binding domain of N. In order to understand the spacial requirements of this boxB/N interaction within the complex, the effects of changes in the length of the nut site linker, the boxB stem, and the peptide spacer connecting the RNA-binding domain and activation domain of N were examined using a bacterial reporter system. As a result, we found that the requirements for the boxB stem length and N peptide linker length were optimized and strict. In contrast, when the boxB/N interaction was replaced by heterologous RNA/peptide interactions, the strict requirement for the length of the peptide linker and the RNA stem was relaxed, presumably due to the absence of the interaction between boxB/N and the host factor NusA in the wild-type complex. It was also shown that the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer, suggesting that a further understanding of the organization of the antitermination complex may provide insights into the engineering of functional ribonucleoprotein complexes.

Replacement of the lambda boxB RNA-N peptide with heterologous RNA-peptide interactions relaxes the strict spatial requirements for the formation of a transcription anti-termination complex.

In bacteriophage lambda, formation of a transcriptional anti-termination complex involving the elongating RNA polymerase is mediated by the interaction of boxB RNA with the RNA-binding domain of the N protein (N peptide). In an attempt to understand the spatial requirements for boxB/N peptide interaction within the anti-termination complex, the effects of changes in the distance between boxA and boxB RNA, the length of the boxB stem, and the distance between the N peptide and remainder of the N protein were examined using a bacterial reporter system. It was found that the requirements for boxB stem length and the distance between N peptide and the remainder of N were optimized and strict. In contrast, replacement of the boxB/N interaction by heterologous RNA-peptide interactions appeared to relax the strict requirement for RNA stem length and the orientation of the RNA-binding peptide, presumably due to the absence of the cooperative interaction between boxB/N and the host factor NusA. In addition, the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer. A further understanding of the structural organization of the anti-termination complex may provide insights into how functional ribonucleoprotein complexes may be engineered.