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.
Horiya
Satoru
S
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Inaba
Mitsuru
M
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Koh
Chang-Song
CS
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Uehara
Hiroaki
H
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Masui
Naomi
N
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Mizuguchi
Misa
M
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Ishibashi
Masaya
M
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Matsufuji
Senya
S
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
Harada
Kazuo
K
Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.Department of Life Sciences, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan.Shinkasoyaku Corp., Yokohama-shi, Kanagawa 240-0064, Japan.
D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018388:Codon, Terminator; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D018388:Codon, Terminator; C059417:N protein, Bacteriophage lambda; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054334:Viral Regulatory and Accessory Proteins