Action of an RNA site at a distance: role of the nut genetic signal in transcription antitermination by phage-lambda N gene product.
The N gene product of Escherichia coli phage lambda is a transcriptional activator that captures the host RNA polymerase and modifies it to a termination-resistant form, permitting gene expression in two large polycistronic operons of the phage genome. Antitermination in vitro requires at least one host factor called NusA, which directly binds the N protein as well as RNA polymerase, and also a transcribed cis-acting site known as nut, within which lies the hypothesized N-recognition signal, boxB. BoxB is an interrupted palindrome capable of forming a hairpin in the mRNA. Inhibition studies with complementary DNA oligonucleotides provide evidence for a direct role of the boxB hairpin in antitermination. Kinetic studies of transcript elongation reveal that the boxB hairpin does not induce an appreciable pause to hold polymerase captive for engagement by N and NusA. Moreover, the efficiency of antitermination remains virtually the same whether N and NusA are added early, prior to nut site transcription, or added later, after the polymerase has already transcribed past the nut site. After transcription of the nut site, RNA polymerase remains susceptible to modification by N and NusA for an appreciable amount of time and distance, and the nut site DNA becomes dispensable for this modification. These results lead to the hypothesis that the boxB RNA hairpin acts in a manner analogous to the DNA enhancers, binding N and mediating a productive polymerase-NusA-N interaction by mRNA looping.
Whalen
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Department of Microbiology, University of Connecticut Health Center, Farmington 06030.
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D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors; D012318:RNA Polymerase I / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; 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; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012367:RNA, Viral; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins; C068727:nusA protein, E coli; D004247:DNA; D012318:RNA Polymerase I