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Two Escherichia coli control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites P1 and P2, both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter. The final complexes were characterized by their topological unwinding, by DNase I and orthophenanthroline footprints, as well as by methylation of unpaired cytosine residues. At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow. The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of nucleotide substrates as well as during an abortive initiation cycle. At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period. Not all the final species are active and unwinding appears to precede, in time, full open-complex formation. At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity. From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing. Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, P1 and P2, on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones). We conclude that negative supercoiling is not an extra-regulatory element of the lactose system, allowing modulation of expression of the wild-type promoter to the profit of P1. Instead, P2 and P1, in the absence of active catabolite receptor protein (CRP-cAMP), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined. The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned. The major effect of CRP-cAMP stimulation appears to be the direct activation at the P1 promoter.
J Mol Biol 1987 Jun 20
PMID:Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. 330 41

The nucleotide sequences of the glpT gene of Escherichia coli and its regulatory region have been elucidated and the primary structure of the glycerol-3-phosphate transport protein deduced. Extensive amino acid sequence homology was found with two other cytoplasmic membrane proteins: the functionally related hexose-6-phosphate transport protein, and the UHPC protein involved in regulating hexose-6-phosphate uptake. Although no significant amino acid sequence homology was found with other transport proteins, such as the arabinose, citrate, glucose, melibiose, lactose or xylose transporters, all of these proteins share a common secondary structure arrangement with the GLP T protein as they apparently contain twelve membrane-spanning alpha-helical segments. The promoter for glpT was located by transcript mapping and shown to overlap a site to which catabolite activator protein binds in vitro. These findings indicate how catabolite repression may be mediated but do not explain its physiological significance in glycerol metabolism.
Mol Microbiol 1987 Nov
PMID:Nucleotide sequence and transcriptional startpoint of the glpT gene of Escherichia coli: extensive sequence homology of the glycerol-3-phosphate transport protein with components of the hexose-6-phosphate transport system. 332 81

New pleiotropic mutants were isolated that express either the phoA, psiE or psiO promoter constitutively and simultaneously alter bacterial alkaline phosphatase regulation, carbon utilization or ultraviolet light sensitivity. To do this, Lac+ mutants were isolated from strains with the appropriate lacZ transcriptional fusions. Over 300 independent mutants were characterized, and all that constitutively express phoA map in phoR, phoU, the phosphate-specific transport system or a new locus called phoF. However, only phoU mutants express both phoA and psiE constitutively. Carbohydrate-utilizing mutants that show constitutive expression of psiE and psiO map in cya, crp and, possibly, crr. Also, numerous ultraviolet-light-sensitive mutants were discovered that show increased psiO expression and map in lon. Some other mutations that lead to constitutive psiO expression (which is normally induced either by phosphate, nitrogen or carbon starvation or anoxia) show decreased expression of phoA. Also, several mutants were found that show an unusual metastable character affecting psiO or phoA transcription. In these, colonies spontaneously switch between an induced and repressed "state" with respect to lac or bacterial alkaline phosphatase expression. In some, the clonal variation of the lactose phenotype or bacterial alkaline phosphatase synthesis is recA-independent and phenotypically resembles phase variation in Salmonella typhimurium. The latter class are called "phase mutants". The mutants are discussed in terms of protein-nucleic acid interactions and/or possible changes in the DNA, i.e. modifications or rearrangements, within the phosphate gene system, that are physiologically regulated.
J Mol Biol 1986 Sep 05
PMID:Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. 354 Mar 12

Covalent monoadducts are the major types of gene damage after exposure to chemical mutagens and carcinogens. These and other damage structures together give rise to the spectrum of mutations that includes base-pair substitutions, insertions and deletions. In this study we introduced the bulky adduct guanine-8-aminofluorene into defined sites on one or both strands of the lactose operator. After insertion into plasmid pBR322 and replication in Escherichia coli COEC40, operator mutants were recognized on 5-bromo,4-chloro,3-indolyl-beta-D-galactoside plates and by hybridization probing. Out of ten randomly selected mutants, nine were single-base deletions and one was a two-base deletion. All mutations were at the site of modification or immediately adjacent to that site. If modifications were placed into both plasmid strands, preventing excision repair, operator mutants comprised close to 100% of operator-containing plasmids.
J Mol Biol 1986 Sep 20
PMID:Mutagenesis originating in site-specific DNA damage. 354 76

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.
Mol Cell Biol 1987 Mar
PMID:Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae. 355 Apr 30

Several reducing sugars with structural analogy (glucose, galactose, lactose, melibiose, maltose and cellobiose) were bound to a carrier protein ovalbumin by amino-carbonyl reaction, and the non-enzymatically glycosylated proteins were injected into mice (BALB/c, C57BL/6, DBA/2 and C3H/He strains) with Freund's adjuvant. Antibody responses to the haptenic sugar antigens were analyzed quantitatively by enzyme-linked immunosorbent-assay using each sugar-bovine serum albumin complex. The haptenic sugar antigen from lactose induced a markedly higher response of specific antibody as compared with the haptenic sugar antigens from the other sugars. The antibody raised against the lactose adduct reacted well with lactulose (4-o-beta-D-galactopyranosyl-D-fructose) or alpha-N-acetyl-epsilon-N-deoxylactulosyl-L-lysine. The results suggested that immunogenicity of haptenic sugar antigens was remarkably different in sugar chemical structure, and that the lactose adduct formed by lactose-protein amino-carbonyl reaction could be an immunodominant antigenic determinant.
Mol Immunol 1987 May
PMID:Antibody response to haptenic sugar antigen: immunodominancy of protein-bound lactose formed by amino-carbonyl reaction. 365 87

The binding specificity of rat alveolar macrophages (AM phi) for sheep erythrocytes (E) coated with gangliosides GM1, GM2, GM3, GD1a, GD1b or GT1b was analyzed in a rosette assay by studying the inhibitory effect of gangliosides, various carbohydrates, IgG, C3b-like C3, and fibronectin in this assay. The uptake of gangliosides by E was calculated from radioactivity measurements using 3H-labeled gangliosides. The different gangliosides were taken up by E at 37 degrees C to a similar extent. Uptake of 3H-labeled GM2 correlated linearly to its concn in the incubation medium. Erythrocytes pretreated with the same molar concn of GM2, GD1a, GD1b or GT1b were bound to AM phi to the same degree reaching a maximum of about 90% rosette forming cells. A mean of 17.8% AM phi-bound GM3-coated E. Treatment of E with asialo-GM2 (GA2) or GM1 did not induce significant rosette formation. A dose-dependent inhibition of rosette formation was observed when AM phi were preincubated at 0 degree C with GM2, GM3, GD1a, GD1b or GT1b, but not with GM1 or GA2 Of the tested carbohydrates, sialyl-lactose had a strong inhibitory effect, while lactose was completely ineffective. N-acetyl-neuraminic acid, N-glycolyl-neuraminic acid and N-acetyl-galactosamine were slightly inhibitory. A series of other carbohydrates including highly negatively charged compounds, as well as fibronectin, IgG or C3b-like C3 did not show significant inhibition. Our data indicate the expression of a receptor on rat AM phi recognizing carbohydrates containing sialic acid at or near the non-reducing terminus.
Mol Immunol 1986 Nov
PMID:Specificity of ganglioside binding to rat macrophages. 382 40

The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine. Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex. Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation. The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins. This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts. Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.
J Mol Biol 1985 May 05
PMID:A mutant lactose repressor with altered inducer and operator binding parameters. 389 17

The sequence of ebgR, the gene that encodes the EBG repressor, was determined. There is 44% DNA sequence identity between ebgR and lacI, the gene that encodes the LAC repressor. There is also 25% identity between the amino acid sequence of lacI and the deduced amino acid sequence of ebgR. The sequence of 596 bp distal to ebgA, the structural gene for EBG beta-galactosidase, was also determined. Within that region there were two sequences, 74 and 100 bp long, that showed 46% and 50% identity, respectively, to sequences in the first 600 bp of lacY, the structural gene for the lactose permease. The organization and direction of transcription of the repressor and structural genes of the two operons are identical. Taken together with the homology between ebgA and lacZ (as demonstrated in the companion article in this issue), this provides strong evidence that the EBG and LAC operons are descended from a common ancestor. The map position of these two operons supports the notion that these operons diverged following a genome duplication event in an ancestor of Escherichia coli.
Mol Biol Evol 1985 Nov
PMID:Sequence of the ebgR gene of Escherichia coli: evidence that the EBG and LAC operons are descended from a common ancestor. 393 8

The gene secY (or prlA) is essential for protein export across the cytoplasmic membrane of Escherichia coli. The protein product of secY has been identified using the gene cloned under the control of the pL promoter of phage lambda in combination with the maxicell system. The protein was found to have some unusual properties. First, it is important not to heat the protein at 100 degrees C in the SDS sample buffer for its subsequent detection by gel electrophoresis. Second, migration of the protein in SDS-polyacrylamide gel electrophoresis is variable depending on the gel compositions. Gels with stronger sieving effect give higher apparent molecular weights. These properties are similar to those of hydrophobic proteins of the cytoplasmic membrane, such as the lactose permease. Finally, a major fraction of the protein synthesized from the overproducing plasmid is degraded rapidly in vivo. The altered protein from the secY24 mutant gene is even more unstable. These results provide information which is basic for the dissection of the protein export machinery of the bacterial cell.
Mol Gen Genet 1984
PMID:Identification of the secY (prlA) gene product involved in protein export in Escherichia coli. 609 91


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