Bipartite Function of a Small RNA Hairpin in Transcription Antitermination in Bacteriophage λ

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 9; pp. 4061 - 4065
• Main Authors: Chattopadhyay, Samit, Garcia-Mena, Jaime, DeVito, Joseph, Wolska, Krystyna, Das, Asis
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 25.04.1995; National Acad Sciences; National Academy of Sciences
• Subjects: Antibodies; Bacteria; Bacterial Proteins - metabolism; Bacteriophage lambda - genetics; Bacteriophage lambda - metabolism; Bacteriophages; Base Sequence; Binding Sites; Biochemistry; Cellular biology; Cloning, Molecular; Cooperation; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli Proteins; Gels; Genes, Viral; Integration host factors; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Nucleotides; Nuts; Oligodeoxyribonucleotides; Oligonucleotides; Peptide Elongation Factors; phage lambda; Plasmids; Promoter Regions, Genetic; Proteins; Ribonucleic acid; RNA; RNA, Viral - biosynthesis; RNA, Viral - chemistry; RNA, Viral - metabolism; Substrate Specificity; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional Elongation Factors; Viral Regulatory and Accessory Proteins - metabolism; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.9.4061

Transcription of downstream genes in the early operons of phage λ requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of λ N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.