Synthesis of the nutL DNA segments and analysis of antitermination and termination functions in coliphage lambda

Abstract

The nut antiterminator sequence, when present between a promoter and a terminator, permits the N-mediated antitermination of transcription in phage λ. The efficiency of nutL was determined by assaying the activity of gene galK placed on a plasmid downstream from the promoter, nutL and terminator modules. As a reference and an estimate of the plasmid copy number, we have used an improved and very reproducible assay for bla activity. Sequences consisting of the 17-bp nutL core flanked by two HindIII cohesive sites were synthesized by the phosphite coupling method, and cloned in proper orientation between the pp promoter of pBR322 and λ gene N followed by the t L1 terminator on a galK-expression plasmid. The antitermination efficiencies for two synthetic 17-bp nutL sequences, one wild type and one point mutant at the base of the nutL stem, are similar but substantially reduced in comparison with the native 25-bp nutL sequence cloned at the same site in the otherwise identical galK-expression plasmid. Multiple tandem insertions of the synthetic 17-bp nutL segment successively increase antitermination efficiency, but also to levels below those of comparable plasmids carrying multiple copies of the native 25-bp nutL sequence. Thus, several specific base pairs in the flanking sequences appear to be important for the efficient nut function. In an inverted orientation the 17-bp nutL sequence has lost its antitermination function. It also lost the termination activity exhibited by inversion of the longer 25-bp and 74-bp native nutL sequences.