Specific destruction of the second lac operator decreases repression of the lac operon in Escherichia coli fivefold

The second operator of the lac operon, located within the 5'-coding region of the lacZ gene, was specifically destroyed by means of oligonucleotide-directed mutagenesis. Eight of its bases were exchanged without altering the wild-type amino acid sequence of beta-galactosidase. The mutation was transferred onto an F'lac+I+O+Z+pro+ episome. We observed a fivefold decrease in repression of beta-galactosidase expression compared to that in the wild-type.     

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.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Lac repressor-operator interaction. 8. Lactose is an anti-inducer of the lac operon

Lactose is shown to be an effective anti-inducer of the lac operon both in vivo and in vitro. When lactose is used as a carbon source, the synthesis of β-galaetosidase in Escheriahia coli is not fully induced. Moreover, lactose is able to partially inhibit induction by isopropyl-(β-d-thiogalactoside in strains synthesizing inactive as well as active β-galactosidase. These effects in vivo are not due to catabolite repression by the glucose derived from lactose. These in vivo results suggest that lactose is acting as an anti-inducer. This is confirmed in vitro by showing that lactose binds to the lac represser and stabilizes the represser-operator complex.     

SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon

SsrA RNA of Escherichia coli, also known as 10Sa RNA or tmRNA, acts both as tRNA and mRNA when ribosomes are paused at the 3' end of an mRNA lacking a stop codon. This process, referred to as trans-translation, leads to the addition of a short peptide tag to the C-terminus of the incomplete nascent polypeptide. The tagged polypeptide is then degraded by C-terminal-specific proteases. Here, we focused on endogenous targets for the SsrA system and on a potential regulatory role of SsrA RNA. First, we show that trans-translation events occur frequently in normally growing E. COLI: cells. More specifically, we report that the lacI mRNA encoding Lac repressor (LacI) is a specific natural target for trans-translation. The binding of LacI to the lac operators results in truncated lacI mRNAs that are, in turn, recognized by the SsrA system. The SsrA-mediated tagging and proteolysis of LacI appears to play a role in cellular adaptation to lactose availability by supporting a rapid induction of lac operon expression.     

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.     

Tight-binding repressors of the lac operon: selection system and in vitro analysis.

The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.