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.     

Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.     

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.     

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.     

Translational frameshift sites within bacteriophage lambda genes rexA and cI

Phage lambda's cI-rexA-rexB operon displays an intriguing example of regulation by an unexplained mechanism of polarity. We have identified three potential -1 translational frameshift sites and present a model for translational frameshift suppression by lambda's CI repressor as a mechanism of regulating operon polarity, implying an additional role for CI self-regulation.     

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.     

Mapping of restriction sites in the attachment site region of bacteriophage lambda

Summary A fine structure map of theEcoRI fragment containing the lambda attachment-site region has been constructed. 38 different restriction endonucleases have been employed and 170 sites located in this fragment. In addition, sites in adjacent regions have been determined for several enzymes. Complete cleavage maps of the entire lambda genome have been obtained for endonucleasesBglII,BluI,KpnI,SacI,SacII,SalI andXbaI. The strategy employed for mapping included comparison of deletion and substitution mutants, analysis of mixed digests, and detailed analysis of subfragments.