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At least 19 recordsLinked to original sources
PubMed1977

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.

Bacterial Proteins↗
PubMed1979

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.

Cysteine↗
PubMed1975

Multiple regulation of nucleoside catabolizing enzymes: regulation of the deo operon by the cytR and deoR gene products.

The protein and repressor nature of two regulatory gene products in E. coli has been demonstrated, employing mutants with either amber or thermosensitive mutations. The regulatory genes are the cytR and the deoR genes, both of which contribute to the regulation of the synthesis of nucleoside catabolizing enzymes. Enzyme levels in strains with concurrent mutations in both regulatory genes are considerably higher than the sum of the levels in strains with a cytR or a deoR mutation alone, indicating a certain co-operativity between the two repressor proteins. The glucose repression of enzyme levels observed in the double regulatory mutant is similar to that found in a cytR mutant, and much more pronounced than the glucose effect in a deoR mutant. A model of the promoter-operator region in the deo operon is proposed.

Cytidine Deaminase↗
PubMed1979

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.

Amino Acids↗
PubMed1979

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.

Amino Acid Sequence↗
PubMedDate not supplied

Transposition protein of Tn3: identification and characterisation of an essential repressor-controlled gene product.

Fusions that bring lac gene expression under the control of transcriptional and translational signals within the Tn3 element have been used to identify and characterise a Tn3-encoded 'transposase' (Tnp) peptide of MW 100,000 essential for transposition. The gene specifying this product is regulated by the Tn3 repressor protein and is part of a bidirectional genetic unit that includes the repressor gene.

Bacterial Proteins↗
PubMed1979

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.

Amino Acids↗
PubMed1978

beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Amino Acid Sequence↗
PubMed1979

Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene.

From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively. The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis. They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression. Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background. Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose. The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex. All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region. The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex.

Adenylyl Cyclases↗
PubMed1977

5'-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: features of translational initiation and reinitiation sites.

In a sequence of 214 nucleotides at the 5' terminus of the I gene mRNA, which codes for the lactose repressor protein of Escherichia coli, (i) an untranslated leader sequence of 28 residues precedes the repressor coding region; (ii) a GUG initiates synthesis of the wild-type repressor; (iii) GUG and AUG are the functional initiators for the synthesis of restart polypeptides activated by early I gene amber mutations, confirming previous assignments for these residues based on protein sequencing data; and (iv) sequences complementary to 16S ribosomal RNA provide stronger potential mRNA.16S rRNA interaction at the wild-type initiation site than at the restart sites. When I mRNA is used to direct the formation of initiation complexes in vitro, ribosomes bind only to the wild-type initiator region.A striking feature of the I mRNA sequence is the presence of a number of in-phase GUGs that have not been observed to serve as initiation signals in vivo in the nonsense mutant strains examined. The selective use of potential initiator triplets in the I mRNA leads to the following conclusions. First, when presented with several neighboring initiator triplets at the wild-type initiator region, ribosomes select the one preceded by the strongest appropriately positioned complementarity to the 16S 3' end. Second, ribosomes do not restart after termination simply by moving to the next available initiator codon. Third, the formation of stable secondary structures predicted for the untranslated I mRNA beyond chain-terminating nonsense mutations may prevent ribosome access to some potential reinitiation sites.

Bacterial Proteins↗
PubMed1978

In vitro synthesis and and regulation of the biotin enzymes of Escherichia coli K-12.

The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system. These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A. Guha, Y. Saturen, and W. Szybalski, J. Mol. Biol. 56:53-62, 1971). The kinetics of synthesis of both the enzymes were determined and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells. Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis. Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene. However, the two strands showed differential repression. At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations. Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators.

Biotin↗