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Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.
Initiation of transcription of the lac operon by RNA polymerase (R) is inhibited by binding of lac repressor (L) to an operator site which overlaps the lac promoter (P). We have investigated repression of the lac UV5 promoter in vitro for a choice of the repressor--operator binding constant and ranges of thermodynamic activities of L and R which appear to be relevant in vivo. Effects of [L] on the extent of formation and the kinetics of association and dissociation of abortively-initiating open complexes (RPinit) were examined using fluorescence detected abortive initiation and KMnO4 chemical probing. The nitrocellulose filter assay was used to measure the dissociation rate constant and the equilibrium constant for binding for L to its operator site in the absence of R. For the chosen solution conditions, we find that both the observed velocity of abortive RNA oligomer synthesis and the KMnO4 reactivities of bases in the open region are functions of [L] and [R], demonstrating that formation of both RPinit and the repressor-operator complex (PL) are reversible processes under these conditions, and requiring the use of a relaxation-to-equilibrium analysis to interpret the kinetics. The agreement between dissociation rate constants of RPinit when challenged with either lac repressor or heparin, and the dependences on [L] and [R] of abortive synthesis velocities at binding equilibrium and of relaxation rate constants for reversible formation of RPinit from PL, all provide evidence for a simple competition mechanism. In this mechanism, and in contrast to recent proposals from other laboratories, lac repressor inhibits formation of RPinit and hence the observed rate of abortive product synthesis by reducing the equilibrium extent of formation of the first closed complex (RPc1), without affecting either the nature of RPinit or steps in formation of RPinit from RPc1.
Silencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS. Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter. However, the expression level of activated bgl operon derivatives varies with the strain background. Here it is shown that the repression of the bgl operon by RpoS requires Crl. Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes. In a Crl-negative strain RpoS had no effect on the bgl operon. The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E. coli strains. Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.
The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products. The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF::Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ::Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ. The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome.
The control of transcription initiation at the lactose operon promoter was investigated in vitro. We found that an upstream promoter (termed lac P2) interfered with RNA polymerase binding at the principal promoter (termed lac P1). The start site for lac P2 was located at base pair position -22 relative to the P1 start site. The addition of cAMP receptor protein and cAMP was shown to repress lac P2 and to activate lac P1. Abortive initiation reactions for both promoters were used to investigate the coordinate repression-activation elicited by CRP-cAMP. The effects of lac promoter mutations (L8, Ps, and UV5) were consistent with an important RNA polymerase positioning role for CRP-cAMP in the activation of lac operon expression.
Mutations that result in small decreases or increases in expression from the Escherichia coli galactose operon promoter region can be detected by using a plasmid in which the gal promoters were fused to the lac operon. We describe how the level of lac expression was adjusted so that the Lac phenotype of host cells was optimally sensitive to changes in the gal promoter sequence. We have investigated the properties of two new gal promoter mutations both in vivo and in vitro, and have determined their effects on the two overlapping gal promoters, P1 and P2. Although one mutation causes only a small reduction in overall expression in vivo, it completely suppresses transcription initiation at the P1 promoter. However, it also increases expression from the P2 promoter, which compensates for the change at P1. This mutation, a GC to AT transition, falls in a zone just upstream of the P1 Pribnow box, which is essential for P1 activity, whilst improving the homology between the P2 Pribnow box and the consensus sequence. The second mutation causes a small increase in P1 activity. This change, a GC to AT transition at -23, falls in the spacer region between the Pribnow box and the -35 region, a zone containing no known promoter consensus sequences. We suggest that this mutation, which creates a stretch of five AT base pairs, acts by increasing the twist angle of the sequences in the spacer region. We argue that the increase in promoter activity is due to this twist changing the relative orientation of the Pribnow box and -35 regions.
We have developed a number of broad-host-range plasmids that allow the expression of the Escherichia coli lac operon from any cloned promoter, and the creation of 'in phase' fusions between lacZ and other cloned genes. In a second series of constructions, the E. coli gal operon has been cloned into the broad-host-range vector and a plasmid carrying both the E. coli gal and lac genes is described. These plasmids have been transferred into Pseudomonas aeruginosa and Zymomonas mobilis and their effects on the utilisation of lactose and galactose have been investigated.
Derivatives of lambda dv whose pRoR promoter was replaced by lactose operon (lac promoter) were constructed and cloned in pBR322 plasmid. They were named pLOP-1, 2, and 3, and their structures are shown in Fig. 1. These plasmids were introduced into Escherichia coli (E. coli) lac iq and the effect of the inducer of lac promoter on the synthesis of plasmid DNA was examined. The synthesis of pLOP-2 or 3 DNA was strongly stimulated. pLOP-1, however, responded poorly to the inducer. Plasmids pLOP-2 and 3 were not segregated evenly into daughter cells after the induction and most of the progeny cells did not receive the plasmid. The synthesis of plasmid DNA after induction depended on the function of O and P genes and was inhibited by the addition of rifampicin and chloramphenicol. Most of the plasmid DNA synthesized after the induction cosedimented with the folded host chromosomal complex, suggesting an unusual structure of the DNA. When the inducer was removed, normal segregation of the plasmid resumed and the copy number of plasmid DNA decreased to the original level.
A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen. The nard mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon. Both other known controls, nitrate induction and autoregulation, were unaffected. It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nard mutation affects the Fnr binding site of the narGHI control region.
We determined the location, activity, and regulation of the promoter of the Lactococcus lactis 8-kb lactose operon (lacABCDFEGX), which encodes the enzymes of the lactose phosphotransferase system and the tagatose 6-phosphate pathway. The lac promoter sequence corresponds closely to the consensus promoter described for gram-positive bacteria and is located in a back-to-back configuration with the promoter of the divergently transcribed lacR gene, which encodes the LacR repressor. The transcription start sites used under induced (lactose) and noninduced (glucose) conditions were determined. The minimal promoter region that could be isolated on a single restriction fragment included sequences ranging from -75 to +42. The effect of the presence of flanking sequences and the lacR gene on promoter activity and regulation was studied in Escherichia coli and L. lactis strains by using transcriptional fusions with promoterless chloramphenicol acetyltransferase reporter genes. The results showed that transcriptional regulation of the lac operon is mediated by the interaction between the LacR repressor, the lac promoter, and sequences in the noncoding region between the lacR and lacA genes. Sequences flanking the minimal promoter region appeared to enhance lac promoter activity much more in L. lactis (5- to 38-fold) than in E. coli (1.3- to 5-fold).
A mathematical model for the regulation of induction in the lac operon in Escherichia coli is presented. This model takes into account the dynamics of the permease facilitating the internalization of external lactose; internal lactose; beta-galactosidase, which is involved in the conversion of lactose to allolactose, glucose and galactose; the allolactose interactions with the lac repressor; and mRNA. The final model consists of five nonlinear differential delay equations with delays due to the transcription and translation process. We have paid particular attention to the estimation of the parameters in the model. We have tested our model against two sets of beta-galactosidase activity versus time data, as well as a set of data on beta-galactosidase activity during periodic phosphate feeding. In all three cases we find excellent agreement between the data and the model predictions. Analytical and numerical studies also indicate that for physiologically realistic values of the external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics.
A reversible block to RNA polymerase II transcriptional elongation has been created with a lac operator sequence in the intron of the SV40 large T-antigen gene. When this transcription unit is injected into rabbit kidney cells expressing Escherichia coli lac repressor, T-antigen expression is reduced. This effect is not observed in cells lacking repressor or in the absence of the operator, and it is reversed by an inducer of the lac operon, namely isopropyl thiogalactoside (IPTG). In an extract of HeLa nuclei supplemented with lac repressor, this and similar constructs give rise to shortened transcripts that map to the 5' boundary of the repressor-operator complex. These shorter RNAs are also sensitive to IPTG induction. This model system shows that a protein-DNA complex can block the passage of RNA polymerase II, and offers some insight into the control of eukaryotic gene expression during transcription elongation, a phenomenon observed in a variety of systems.
cya-lac and crp-lac operon fusions were isolated in Salmonella typhimurium by using the phage Mu d1(lac cts Apr). Both transduction and reversion analyses have indicated that lac expression is controlled by the appropriate promoter, e.g., either crpp or cyap. By using chromosomal mobilization techniques, we found that cya had a clockwise direction of transcription on the standard S. typhimurium map. The cya-lac fusions could be complemented by Escherichia coli F'133, which covers cya, with a resultant 17 to 38% decrease in cya expression. Cyclic AMP was found to be able to repress the expression of the cya-lac fusion ninefold when present at 25 mM. This repression was not seen in crp backgrounds, and hence is mediated by the cAMP receptor protein. Repression of cya was also found upon growth on carbon sources known to elicit high cyclic AMP levels.
A broad-host-range vector system for studying translational fusions was constructed. The region that retains the origin of replication, nic, mob, and rep genes of the broad-host-range plasmid RSF1010 was isolated as either an HincII or a PstI-PvuII restriction fragment. These restriction fragments were ligated to tetracycline, kanamycin, or streptomycin/spectinomycin resistance genes to generate plasmids pUI501, pUI511, pUI504, and pUI506. A functional lacZ gene lacking downstream lac operon sequences together with the lac promoter was constructed from plasmids pMC1871 and pUC18. This lacZ gene was inserted into pUI501 and pUI511 to generate plasmids pUI502, pUI503, pUI512, and pUI513. An oligodeoxynucleotide sequence that carries three unique blunt-end restriction sites was synthesized, annealed, and ligated in frame to the amino-terminal end of the lacZ gene in each of these plasmids. This multiple cloning sequence will allow translational fusions to the lacZ gene in all three reading frames. The stability of these plasmids and the expression of the lacZ gene in both Escherichia coli and Rhodobacter sphaeroides were studied.
Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.
Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.
The Escherichia coli lac operon provides a classic paradigm for understanding regulation of gene transcription. It is now appreciated that lac promoter repression involves cooperative binding of the bidentate lac repressor tetramer to pairs of lac operators via DNA looping. We have adapted components of this system to create an artificial assay of DNA flexibility in E.coli. This approach allows for systematic study of endogenous and exogenous proteins as architectural factors that enhance apparent DNA flexibility in vivo. We show that inducer binding does not completely remove repression loops but it does alter their geometries. Deletion of the E.coli HU protein drastically destabilizes small repression loops, an effect that can be partially overcome by expression of a heterologous mammalian HMG protein. These results emphasize that the inherent torsional inflexibility of DNA restrains looping and must be modulated in vivo.
The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.