title: Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements.
abstract: 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.
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005814:Genes, Viral; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes