trp repressor interactions with the trp aroH and trpR operators. Comparison of repressor binding in vitro and repression in vivo

Interaction of the Escherichia coli trp repressor with the promoter-operator regions of the trp, aroH and trpR operons was studied in vivo and in vitro. The three operators have similar, but non-identical, sequences; each operator is located in a different segment of its respective promoter. In vivo repression of the three operons was measured using single-copy gene fusions to lacZ. The extent of repression varied from 300-fold for the trp operon, to sixfold for the aroH operon and threefold for the trpR operon. To determine whether differential binding of repressor to the three operators was responsible for the differences in repression observed in vivo, three in vitro binding assays were employed. Restriction-site protection, gel retardation and DNase footprinting analyses revealed that repressor binds to the three operators with almost equal affinity. It was also shown in an in vivo competition assay that repressor binds approximately equally well to each of the three operators. It is proposed that the differential regulation observed in vivo may be due to the different relative locations of the three operators within their respective promoters.     

Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae.     

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.     

Studies on the interaction of Trp holorepressor with several operators. Evidence that the target need not be palindromic

The interaction of Trp repressor protein with partial trp operators was studied in vitro and in vivo. At high ratios of protein to DNA, Trp holorepressor formed stable complexes with DNA molecules containing half operators. When plasmids conferring the capacity to hyperproduce Trp repressor were present in trpOc strains of Escherichia coli, repression of downstream tryptophan synthase occurred. Palindromicity of the trp operator may facilitate stable interaction with Trp repressor, but this attribute need not be regarded as a critically essential structural feature. Sufficient information for the recognition by Trp repressor protein of an appropriate target resides within a DNA sequence of approximately ten base-pairs.     

Intracellular Trp repressor levels in Escherichia coli.

A radioimmunoassay for the Trp repressor protein of Escherichia coli was developed with antisera raised against purified Trp repressor protein. This assay was used to directly measure the intracellular Trp repressor content in several E. coli K-12 and B/r strains. Repressor levels varied from 2.5- to 3-fold in response to L-tryptophan concentration in the growth medium (15 to 44 ng of repressor per mg of protein). Neither cell growth rate nor culture age had a significant effect on repressor concentrations within the cell. Addition of L-tryptophan to the growth medium resulted in lowered intracellular levels of Trp repressor. The absolute amounts of native Trp repressor molecules per cell varied between 120 and 375 dimers in the presence and absence of L-tryptophan in the culture medium, respectively. Assuming an intracellular volume of 7.3 microliters/10(10) E. coli cells, the Trp repressor concentration varied from 270 to 850 nM in response to extracellular tryptophan levels. These findings represent the first direct measurements of Trp repressor levels in E. coli and confirm the autoregulatory nature of the trpR gene.     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

Regulation of tryptophan synthase gene expression in Chlamydia trachomatis

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae-infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan-depleted medium or in the presence of IFN-gamma. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5-fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth-inhibitory properties of 5-fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN-gamma is via the induction of the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase.     

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.     

Temperature-sensitive repression of the tryptophan operon in Escherichia coli

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.     

Location of trpR Mutations in the serB-thr Region of Salmonella typhimurium

Tryptophan biosynthesis in Salmonella is controlled by at least one regulatory gene, trpR, which is cotransducible with thr genes and not with the trp operon. Mutations in trpR cause derepression of tryptophan enzyme synthesis and confer resistance to growth inhibition by 5-methyltryptophan. Nineteen trpR mutations were mapped with respect to thrA and serB markers by two-point (ratio) and three-point transduction tests. The results are all consistent with the site order serB80-trpR-thrA59 on the Salmonella chromosome. Very low or undetectable levels of recombination between different trpR mutations have so far prevented the determination of fine structure in the trpR gene. Thirteen other 5-methyltryptophan-resistant mutants previously found not to be cotransducible with either the trp operon or thrA, and designated trpT, were also used in these experiments. Lack of cotransducibility with thrA was confirmed, and no linkage with serB was detected. The nature and location of trpT mutations remain obscure.     

DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor.

A DNA sequence of 1041 base pairs from a BamHI fragment containing the E. coli trpR gene has been determined. With this sequence and other experimental evidence, the primary structure (88 amino acids) of the Trp repressor can be predicted. Additional features of the DNA sequences include a 22 base pair region upstream from the proposed structural gene which exhibits striking homology with the trp operator, thus implying that expression of the trpR gene may be under autogenous regulation.