Genetic fusions that help define a transcription termination region in Escherichia coli

The trpA gene product was analyzed from a class of strains of Escherichia coli K12 in which the lac operon has been fused by deletion to the trp operon. These are strains that have retained the ability to synthesize tryptophan. Two of these strains are shown to make a wild-type trpA product; these strains retain intact all structural genes of the ttrp operon. It is proposed that the lac operon in these strains is fused to a region of the trp operon between trpA, the last gene in the operon, and the region where trp messenger RNA synthesis terminates. The region where trp messenger RNA synthesis terminates thus is distinct from the trp structural genes.     

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

Summary Two sets of plaque-forming transducing phages have been isolated which carry parts of the tryptophan (trp) and lactose (lac) operons. The ptrp-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 ptrp/lac, carry trp and lac in opposing orientations. These ptrp/lac phages contain deletions which remove all of the lac structural genes and end between the operator and the z gene.A preliminary report of a portion of this work was presented at the Thirteenth International Congress of Genetics, Berkeley, 1973.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

Re-initiation of tryptophan operon expression in a promoter deletion strain of Salmonella typhimurium.

Summary A genetic and enzymological study was made of five spontaneous prototrophic revertants of a tryptophan auxotroph of Salmonella typhimurium which carries a deletion extending from the closely linked supX locus into the trp operator-promoter region. The revertants were found to have regained initiation of expression of all five trp genes. Recombinational tests showed that in each case the genetic change responsible for re-initiation is cotransducible with the trp-cysB region of the chromosome. Two different mechanisms leading to re-initiation of trp gene expression were established: (a) an extension of the limits of the original deletion resulting in the fusion of the trp structural genes with a nearby gene or gene set located outside the operator end of trp, and (b) translocation of a duplicate set of the trp structural genes to other chromosomal sites, located operator-distal to the normal trp operon, in such a manner that they are functionally fused to foreign genetic units. One revertant which arose by mechanism (a) was shown to have an extended deletion with one new terminus in trp and the other in the nearby cysB locus. All the revertants exhibit constitutive expression of the trp enzymes, with activities varying among strains from five to forty five times greater than the fully repressed wild type level. The protein product of trpA, the first structural gene of the operon, appears to have been partially damaged by the re-initiation event in at least two strains, while in the other strains, the enzyme appears in preliminary tests to be indistinguishable from that of wild type.     

Direct identification of the amino acid changes in two mutant lac repressors.

Deletions extending into the trp operon at one terminus and the lacI control region at the other terminus have been examined. One of these, B116, ends within the trp leader sequence and eliminates the trp attenuator site, placing the synthesis of lac repressor under trp control. We have isolated and characterized the B116 repressor. The protein sequence of the aminoterminus of B116 shows that an additional 16 residues are added to the amino-terminal end of wild-type repressor. Moreover, a valine residue appears in place of methionine at position 17 (the original amino-terminal residue of the wild-type repressor). A comparison of the messenger RNA sequence of the trp leader region and of the I leader region demonstrates that the translation of the B116 repressor is initiated at an AUG codon within the trp leader sequence. The GUG initiation codon at the start point for translation of wild-type repressor is now read as valine, since it appears at an internal position (residue 17 of the altered repressor). The B116 repressor accumulates at levels as high as 1% of the soluble cell protein in trpR^? strains. The efficiency of the trp leader initiation codon in translation suggests that in wild-type strains this AUG is also active in directing protein synthesis, which would result in a polypeptide consisting of 14 amino acids. We have examined the physical properties of the B116 repressor, which shows a marked tendency to form higher aggregates. Other characteristics of B116 are also described.     

Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function

Among a collection of 34 independent mutants with internal deletions in the trp operon of Escherichia coli we found six that fail to recombine with any known point mutant in trpE, the first gene in the operon. These six deletion mutants are regulated normally by tryptophan and thus appear to have the trp operator region intact. However, four of these deletions result in alterations in the maximum level of expression of the trpC, B and A genes when compared with wild type or with an internal deletion of similar length which retains a small operatorproximal segment of trpE. Two of these deletion mutants, trpΔED1 and trpΔED12, have lower levels of the protein products of trpB and trpA than the control strains. In contrast, deletions trpΔED2 and trpΔED102 both markedly increase the levels of the trpB and trpA polypeptides. Deletion mutant trpΔED2 has 3 to 3.5 times and mutant trpΔED102 has seven to eight times as much tryptophan synthetase β₂ and α proteins as the wild-type or deletion control strains. The increase in tryptophan synthetase β₂ and α proteins seen is a consequence of an increase in the level of trp mRNA directing the synthesis of these enzymes. The rate of synthesis of trpBA mRNA is increased in trpΔAED2 about twofold, and in trpΔED102 about four- to sixfold over the control strain. The left-hand deletion end-points of both trpΔED2 and trpΔAED102 have been shown to map to the right of a known trp operator-constitutive mutation and appear to lie before the first translation start codon in trpE (M. Bronson, C. Squires &; C. Yanofsky, unpublished results). We propose that these deletions alter a region between the earliest known trpE point mutation and the trp operator which influences the maximum rate of synthesis of trp operon mRNA.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change

The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons.     

Simple Measurement of Blood NADP and Blood Levels of NAD and NADP in Humans

The tryptophan synthase genes, trpA and trpB, of Bacillus stearothermophilus IFO13737 were cloned by transformation of tryptophan auxotrophic mutations of the trp genes into Escherichia coli. The genes are located in the order of trpB and trp A, according to their coding orientation, in a 2.5 kb EcoRy-Hindlll DNA fragment. The complete nucleotide sequence of this DNA was determined. The trp A and trpB genes consist of 810bp (269 amino acid residues) and 1215bp (404 amino acid residues), respectively. The 5′-proximal portion of the trpB gene was found to overlap 20 nucleotides of the upstream coding region of the trpA gene. The homology of the amino acid sequences of the trp gene products of trp A and trpB of B. stearothermophilus is 35 and 50 %, respectively, to those of E. coli, and 55 and 70 %, respectively, to those of B. subtilis.     

Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues

The complete results of the analysis of over 5300 independently derived nonsense mutations in the lacI gene are presented. These have been mapped and divided into specific sites. A total of 105 nonsense mutations derived from 90 different codons can be distinguished, of which several are the result of tandem double base changes induced by ultraviolet light. With the aid of results determined in a preceding paper (Miller et al., 1977), the majority of these mutations have been assigned to points in the gene coding for specific residues in the lac repressor. This allows a detailed correlation of the physical and genetic map.Recombination studies have been carried out using mutations at known sites. For crosses involving mutations separated by less than 30 nucleotides (the main object of this study), a significant lack of agreement between distance and recombination frequency has been found.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.