lac Up-promoter mutants with increased homology to the consensus promoter sequence.

Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.     

Fusion of the promoter region of rRNA operon rrnB to lac Z gene

A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.     

Catabolite repression of the lac operon. Effect of mutations in the lac promoter

1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2-6% of the fully induced wild-type. Each diploid harbours the episome F'lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for beta-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the beta-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of beta-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of beta-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.     

Spontaneous insertion of an IS2 element into the promoter region of the lac operon

A spontaneous mutation in pUC18 has revealed the insertion of a chromosomal insertion sequence (IS)2 element into the promoter region of the lac operon. The IS2 insertion site, at the pentanucleotide sequence TCGAG, is unlike previously described junctional sequences.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Transient repression of the lac operon - the effect of a lac promoter deletion

Experiments have been done to show whether the lac promoter delection L1, which partly alleviates catabolite repression, also affects transient repression of lac. In stain L1/F'M15 all of the beta-galactosidase is synthesized from a chromosomal gene cis to L1, whereas 98% of the thiogalactosidase transacetylase is synthesized from an episomal gene cis to an intact i-p-o region. The addition of glucose to induced cultures of strain L1/F'M15 growing in glycerol medium caused extensive transient repression of transacetylase but almost no transient repression of beta-galactosidase. In control experiments with a diploid stain of genotype p(+)z(+)a(-)/F'p(+)z(-)a(+) the two enzymes suffered equal transient repression. Thus L1 substantially relieves transient repression.     

Protein-DNA cross-linking at the lac promoter.

We report the results of photo-cross-linking of RNA polymerase and the cyclic AMP receptor protein (CRP) to the lac UV5 promoter region carried on either a linear fragment or a supercoiled plasmid. We have devised a protocol that allows the localisation of bases in contact with the protein. RNA polymerase makes contacts within the -10 and -35 regions of the promoter, essentially on the non-template strand. The CRP contact points found in a binary complex are affected by the formation of the ternary complex containing RNA polymerase. Supercoiling has no effect on the position of contacts in any of the complexes. These conclusions were derived from experiments performed using a generally applicable, non-interfering technique that reveals direct contacts between proteins and nucleic acids in nucleoprotein complexes.     

Base substitution mutants of the lac operator: in vivo and in vitro affinities for lac repressor

16 single-site mutations and a 1-bp deletion in the lac operator have been cloned and examined with regard to repressor binding. A 13-bp, central ‘core’ operator sequence, bp 5–17 of the natural operator, was also synthesized and cloned. Repressor affinity was assessed in vivo by quantitating the level of β-galactosidase activity resulting from chromosomal operon derepression and in vitro by measuring the stability of repressor-operator complexes. Our results support the general conclusion that the repressor-operator interaction is asymmetric, particularly across the center of the operator sequence, with little or no specific contact at position 12. Some sequence changes in the right side of the operator markedly reduced repressor affinity, indicating that although binding to this half of the sequence has been suggested to be less important than the left half, it still significantly contributes to the binding affinity.     

Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter.

Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter. They were tested in vivo for function in the presence and absence of lac repressor. We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene. When lac operators are inserted at both sites, we found a greater than 150-fold repression. The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators.     

Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the -35 region was constructed. The mutants have the spacer length increased by five (I5 mutant), or eleven (I11) residues or decreased by eleven residues (D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the -35 region of the gal promoter are fused to the lac -10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal::lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the -35 and -10 regions cannot be easily exchanged between promoters.     

Gene amplification in the lac region of E. coli

We have characterized strains of E. coli in which the lac region, together with varying amounts of surrounding DNA, is amplified 40 to 200 fold. The amplification events involve regions of 7 to 37 kb and result in a tandem array of repeated units. Restriction digest patterns of DNA from over 100 independent strains reveal that the amplified units are different in each case. Mechanisms of gene duplication and amplification, and the relationship of gene amplification in bacteria to that in eucaryotic cells, are considered.