Lac Regulatory Region Research Articles

Adaptive mutation in Escherichia coli.

In 1988 John Cairns, Julie Overbaugh, and Stephan Miller published a paper entitled “The origin of mutants,” in which they suggested that bacteria could choose which mutations to make (11). Shortly thereafter, Cairns and I began collaborating on a National Science Foundation-funded project to investigate the genetic basis of what was popularly called “directed mutation” (although at the time we were calling it “selection-dependent mutation,” which now seems as good a name as any). Our plan was to mutagenize an appropriate strain of Escherichia coli and identify and characterize variants defective in selection-dependent mutation. Cairns and coworkers had been investigating a strain called SM195, described in their original paper (11). However, the mechanisms of mutation in this strain had proved to be complicated, and we wanted a new experimental subject. We were setting up to investigate selection-induced activation of the cryptic bgl operon (36), when Jeffrey Miller gave us a Lac− strain, called α45, that he said had a high rate of reversion to Lac+ after plating on minimum lactose medium. The lac allele in this strain is a fusion of lacI to lacZ that eliminates the lac regulatory region as well as several residues of lacI and lacZ; transcription of the fusion is constitutive, initiating at the lacI promoter (7). α45 has a +1 frameshift mutation, lacI33, in the lacI coding region (12). Miller and coworkers have used several similar strains to study various mutational mechanisms (52, 67). As in many of the strains that originated with Jacob and Monod, proAB and lac are deleted from the chromosome and carried on an episome, F′128. This arrangement greatly facilitates genetic manipulations and turned out to be important to adaptive mutation. We mated the episome from α45 into a Δ(lac-pro) recipient that we had made rifampin resistant and named the new strain FC40 (Foster and Cairns #40).

Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements.

Micrococcal nuclease as a probe for bound and distorted DNA in lac transcription and repression complexes.

Micrococcal nuclease (MNase) is used to probe the structure of transcription and repression complexes at the lac regulatory region in vitro. Both the lac operator, 01, and the pseudo-operator, 03, are found to be protected from MNase digestion by the lac repressor on supercoiled DNA, and hypersensitive sites appear on both strands around nucleotide (nt) -26 between 01 and 03. This hyperreactive site is coincident with the site of the DNA kink shown previously to form within a loop caused by simultaneous repressor binding to 01 and 03. MNase hypersites are also observed both upstream from cAMP receptor protein (CRP) and downstream from bound RNA polymerase in open promoter complexes. In both open and closed complexes the binding of polymerase partially protects the backbone from MNase attack. Catabolite activator protein is shown to be required for both closed and open complex formation. Taken together with previous footprinting data, the results suggest that lac transcription complexes involve DNA bent towards a protein core consisting of RNA polymerase and catabolite activator protein.

Probing the sequence-specific interaction of the cyclic AMP receptor protein with DNA by site-directed mutagenesis.

Mutants in the DNA-binding helix of the cyclic AMP receptor protein (CRP), as well as mutants in a synthetic DNA-binding site derived from the sequence in the lac regulatory region, have been constructed by oligonucleotide-directed mutagenesis, and used to study the effect of selected amino acid substitutions on CRP-mediated transcriptional activity and on sequence-specific DNA binding. It has been shown that mutation of Arg-180 to Lys or Leu abolishes both CRP-mediated expression of beta-galactosidase in vivo and CRP binding of DNA as measured by immunoprecipitation. In contrast, the mutation of Arg-185 to Leu or Lys and the mutation of Lys-188 to Leu does not appear to influence these two parameters significantly. On the DNA side, both substitutions studied, namely the exchange of the G . C base pair in position 2 of the consensus T1G2T3G4A5 motif into an A . T base pair and the exchange of the A . T base pair in position 5 for a G . C base pair, abolish specific binding. Implications of these findings with respect to the present models for specific CRP-DNA recognition are discussed.

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion.

The Escherichia coli mutator gene mutT, which causes A:T----C:G transversion, was cloned in pBR 322. mutT+ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.

Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA.

When the alkB gene of Escherichia coli that controls sensitivity of bacteria to methyl methanesulfonate was placed under the control of the lac regulatory region on a multicopy plasmid, the gene product, AlkB protein, was overproduced. By monitoring the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was purified to near physical homogeneity. An amino-terminal sequence and total amino acid composition of the purified AlkB protein were in accord with the amino acid sequence deduced from the nucleotide sequence of the alkB gene, determined by the phage M13 dideoxy method. It was concluded that the AlkB protein is comprised of 216 amino acids and has a molecular weight of 23,900. The nucleotide sequence analysis also revealed that the ada and alkB genes are adjacent on the E. coli chromosome and that the first initiation codon for AlkB protein overlaps with the termination codon for Ada protein. We constructed hybrid plasmids carrying an alkB'-lacZ' fusion, with or without the ada control region, and investigated expression of the alkB gene in response to the alkylating agent. We obtained evidence that the ada and alkB genes constitute an operon.

Regulatory region of the divergent Klebsiella pneumoniae lac operon.

The chromosomal DNA that lies between the lacI and lacZ genes of Klebsiella pneumoniae constitutes a 196-base pair intercistronic region that contains regulatory sequences for both genes. The probable locations of specific regulatory elements for both lacI and lacZ genes were determined by analogy with the corresponding Escherichia coli sequences. A recombinational event in ancestral DNA evidently has inverted the transcriptional direction of lacI in K. pneumoniae relative to the transcriptional direction of lacI in E. coli. One end of the inversion was located within a 19-base pair sequence in the K. pneumoniae regulatory region. Sequences partially homologous to these 19 base pairs were found in two locations on either side of the E. coli lacI gene. The nucleotide sequence of the lac regulatory region in K. pneumoniae exhibits more than one possibility for folded tertiary structures. The spatial relationships of transcriptional binding sites differ in two possible structures. Associations of regulatory and transcriptional proteins with the DNA might affect conformation of the regulatory sequences and, as a consequence, transcription of the lac genes.

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.

LacZ translation initiation mutations

Sixteen single point mutations near the beginning of the lacZ gene have been isolated and their effect on lacZ expression has been measured. Five mutations were obtained that alter a potential stem-and-loop structure in the messenger RNA that masks the initiation codons. Formation of this stem-and-loop is a result of transcription of DNA sequences introduced during the cloning of the lac regulatory region. The mutations isolated were then moved into a background that deleted this structure. Analysis of these mutations indicated that the secondary structure inhibited lacZ expression 5.8-fold and that either single point mutations or a 9 base-pair deletion could relieve this inhibition completely. In addition, it was found that an A to C transversion in the first base following the initiation codon (in the absence of the inhibitory secondary structure) decreases lacZ expression almost twofold, whereas C to U transitions in the next two positions have negligible effects. Mutations were also obtained that either increase or decrease the length of the Shine-Dalgarno sequence. The effects of these mutations were studied in the presence or absence of the secondary structure that involves the two initiation codons. It was found that when translation initiation was inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence increased lacZ expression 2.8-fold and decreasing the length of this sequence reduced lacZ expression 12-fold. When translation initiation was not inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence had no effect and decreasing the length of this sequence only reduced lacZ expression sixfold. The mechanistic implications of these results are discussed. Two initiation codons are located in the beginning of the lacZ gene, 7 and 13 bases from the Shine-Dalgarno sequence. NH 2-terminal sequence analysis indicated that the majority of the protein synthesized initiate at the first initiation codon in the wild-type lacZ gene (in agreement with results reported previously by J. L. Brown and his colleagues). Upon introduction of sequences that result in a change in the mRNA secondary structure, both initiation codons are used in almost equal amounts. Three mutations and two pseudorevertants were obtained, which are located in the first initiation codon. It was found that when the first initiation codon is changed from AUG to GUG, translation initiation is decreased tenfold at that codon. Other mutations at the first initiation codon resulted in initiation occurring exclusively at the second initiation codon at an efficiency that is determined by the nucleotides located in the spacer region.

Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation.

Transcription antitermination by lambda N gene product is affected in a mutant Escherichia coli with altered ribosomal protein S10, caused by the nusE71 mutation. To study the role of translation in antitermination, we have fused the phage nutR locus, the site of action of N, with the lac regulatory region. We have monitored N action by measuring galactokinase, whose synthesis depends on suppression of terminators located between nutR and the galK cistron. We show that a deletion removing potential ribosome binding signals and AUG codons from the upstream region of nut site does not affect N action. Moreover, the lack of translation upstream of nutR does not overcome the antitermination defect caused by nusE mutation. When the upstream region is translated, however, N action is impaired if translation terminates 19 base pairs upstream of nutR . Termination of translation at further upstream sites, such as 23 or 97 base pairs upstream, does not interfere with N action. Our results suggest that the S10 ribosomal protein is required for N action without involving translation. These results also suggest that the nut site RNA itself plays an important role in the formation of a termination-resistant transcription complex.

Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes.

Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region.

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.

Synthesis of a chicken ovalbumin-like protein in the yeast Saccharomyces cerevisiae

A cDNA sequence coding for chicken ovalbumin was fused to the beginning of the Escherichia coli lactose operon and recombined in vitro with a composite vector plasmid which can be propagated in both E. coli and Saccharomyces cerevisiae. Such plasmids direct the synthesis of an ovalbumin-like protein (OLP) in both microorganisms, probably because the E. coli lac regulatory region has some promoter activity in yeast. The yeast strains produce about 1000 to 5000 molecules of ovalbumin-like protein per cell.

“Second” and “third operator” of the lac operon: An investigation of their role in the regulatory mechanism

The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λ dlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under in vivo conditions.

Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro.

A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information for the alpha peptide of beta-galactosidase (beta-D-galactosidegalactohydrolase, EC. 3.2.1.23) has been inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac alpha function. Further characterization of the hybrid phage includes retransformation studies, agarose gel electrophoresis, DNA-DNA hybridization, and heteroduplex mapping. The insertion point has been localized at 0.083 map unit on thewild-type circular map-i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.

Isolation of a functional lac regulatory region.

A DNA fragment containing the lac regulatory region has been isolated by digestion of λ plac 5 DNA with restriction endonuclease from Hemophilus influenzae Rd followed by gel electrophoresis. This DNA fragment, which is approximately 660 base pairs, is identified by its ability to bind purified lac repressor, and has been shown to be functional in a coupled transcription-translation system. A smaller repressor binding fragment of approximately 174 base pairs has been derived from the larger fragment utilizing a secondary digestion with restriction endonuclease from Hemophilus aegyptius.

The construction of lambda transducing phages containing deletions defining regulatory elements of the lac and trp operons in E. coli.

Two sets of plaque-forming λ transducing phages have been isolated which carry parts of the tryptophan (trp) and lactose (lac) operons. The λptrp-lac set of phages carry the trp and lac operons in the same orientation connected by deletions which enter the lac regulatory region from the i side. These deletions start at various sites in or near the trp operon and end either late in the lac i gene, within the promoter, between the promoter and the operator, within the operator, between the operator and the z gene, or very early in the z gene. Starting with one particular trp-lac fusion strain, a series of transducing phages were isolated which contain varying portions of the trp operon extending from the trp A gene towards the trp operator. The other set of phages, which are designated λptrp/lac, carry trp and lac in opposing orientations. These λptrp/lac phages contain deletions which remove all of the lac structural genes and end between the operator and the z gene.