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.
Horiya
Satoru
S
Department of Molecular Biology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
Inaba
Mitsuru
M
Koh
Chang-Song
CS
Uehara
Hiroaki
H
Masui
Naomi
N
Ishibashi
Masaya
M
Matsufuji
Senya
S
Harada
Kazuo
K
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000378:metabolism
C059417:N protein, Bacteriophage lambda; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054334:Viral Regulatory and Accessory Proteins