Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements.
An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005814:Genes, Viral; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D014764:Viral Proteins