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.     

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.     

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module

We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda

For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al. (1981). The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts. Is is also active in vitro, both in the presence or absence of the rho factor. The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo. We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda. The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA. The termination point that follows the last U was determined by the S1 mapping technique.     

Cloning and characterization of the natural lactose operator

A 55-bp DNA segment carrying the wild-type lactose operator sequence has been cloned. Its sequence is: (Formula: see text). With the exceptions of the bases at positions 19 and 41, 26 and 34, and 28 and 32, the sequence is a perfect inverted repeat about base pair 30. This segment was obtained from the wild-type lactose promoter and operator region of lambda h80dlac phage DNA by a combination of in vitro and in vivo steps. Up to four direct-repeat copies of this segment have been cloned in plasmid pMB9 and pBR325. Repressor affinity for this 55-bp fragment does not differ significantly from that for a 40-bp synthetic operator fragment cloned previously, even though the 55-bp fragment contains the complete set of sequence symmetries associated with the natural operator, whereas the 40-bp fragment does not. An improved procedure for operator purification is described: this was used to prepare 14 mg of the 55-bp fragment over a 2-month period.