Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.
Transcription of downstream genes in the early operons of phage lambda 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 lambda 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.
Chattopadhyay
S
S
Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA.
Garcia-Mena
J
J
DeVito
J
J
Wolska
K
K
Das
A
A
D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005814:Genes, Viral; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010445:Peptide Elongation Factors; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C059417:N protein, Bacteriophage lambda; D009838:Oligodeoxyribonucleotides; D010445:Peptide Elongation Factors; D012367:RNA, Viral; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases