Relief of polarity in DNA-dependent cell-free synthesis of enzymes of the galactose operon of Escherichia coli

Summary Polar mutations of the galactose operon of both, nonsense and insertion type have been studied in a system for DNA-dependent synthesis of the galactose enzymes of Escherichia coli. In vivo, these mutations reduce to different degrees the level of expression of the gene located on the promoter-distal side of the mutation. No such polar effects are observed in vitro. This relief of polarity is neither due to the action of nonsense suppressors, nor to random initiation of mRNA synthesis.A special aspect of this study concerns those insertion mutations which carry a segment of DNA of foreign origin inserted near the control region of the galactose operon. In vivo, mutants of this type produce only one percent or less of the three galactose enzymes as compared to the wildtype. The residual enzyme synthesis is not or only slightly affected by inducer. In contrast, DNA carrying such insertion mutations is fully active in the cell-free enzyme synthesis and sensitive to the controls exerted by the galactose repressor and by the catabolite gene activator protein.     

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 Genetic Basis for Mucoidy and Radiation Sensitivity in capR (lon) Mutants of E. coli K-12

CapR mutants of E. coli K-12 overproduce capsular polysaccharide (mucoid phenotype) and enzymes involved in capsular polysaccharide synthesis, and they are sensitive to radiation. It has been uncertain whether both properties are mediated by damage to a single cistron or by a polar effect on a second cistron in the same operon. Introduction of a polarity suppressor caused no change in the overproduction of polysaccharide, in the enzymes of polysaccharide synthesis or in radiation sensitivity of the capR mutant. Thus mucoidy and radiation sensitivity resulting from capR (lon) mutations are both the consequences of impairment of the same cistron. The experiments demonstrate the advantage of the use of polarity suppressors (over conventional nonsense suppressors) in determining whether pleiotropic effects of a mutation are the result of polarity.     

Expression of the tryptophan operon in merodiploids of Escherichia coli. I. Gene dosage, gene position and marker effects

Summary Under conditions of derepression,Escherichia coli K12 strains diploid for thetrp operon specify more than twice as much enzyme as a haploid. The disproportionate increase probably occurs because episomally carriedtrp genes tend to specify more enzyme than do chromosomal genes.Operons harboring the nonsense mutationtrpA2 or the missense mutationtrpBYS-101 specify less protein than do wild-type operons. This effect varies with operon location in the case oftrpBYS-101.In a homozygoustrp merodiploid A46/F A46 reversion totrp ⁺ occurs three times as frequently in episomal DNA as in chromosomal DNA. Thus, if the chromosome: Ftrp episome ratio inE. coli is one, as demonstrated by Helinski and co-workers, the rate of gene expression and the rate of mutation can vary and depends upon the location of the DNA within the cell.Supported by Grant AM-12150 from the National Institutes of Health. Journal Paper No. 3973 of Purdue Agricultural Experiment Station.     

Genetic fusions defining trp and lac operon regulatory elements

Two procedures for easily isolating deletions that fuse the trp and lac operons are described. Using these procedures, a large number of fusion deletions have been isolated. The lac ends of these deletions extend varying distances into the lacI gene and the lac promoter-operator region. Therefore, contrary to a previous report, there does not appear to be a messenger-termination signal at the C-terminal end of the lacI gene.The trp ends of fusion deletions do not have to extend into the trp structural genes to effect fusion, suggesting that mRNA synthesis initiated at the trp promoter proceeds some distance beyond the trp structural genes before a messenger termination signal is reached. Deletions that extend a short distance into the C-terminus of trpA, the last gene in the trp operon, do not completely abolish activity of the trpA product.The procedures described for isolating fusions of the trp and lac operons can be generalized to other systems.     

Strong-polar mutations in the transferase gene of the galactose operon in E.coli

Summary A class of mutations in the transferase gene of the galactose operon in E. coli is described, which is strongly polar for the synthesis of kinase. The latter enzyme is made only to the extent of about 0.1% of the amount made in the induced wildtype. This amount is not dependent on the map position of the mutations and the residual synthesis is non-inducible. The mutants thus resemble 0° mutants in the same operon.Epimerase, which is coded for by the gene proximal to the transferase gene with respect to the operator, is made in normal amounts and its synthesis is normally inducible.The mutants do not seem to belong either to the nonsense or to the frameshift class on the basis of reversion pattern, suppressibility, and degree of polarity. The possible nature of the mutations is discussed.     

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.