Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wild-type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N-terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.     

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.     

Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors

We have altered the amino acid sequence of the lac repressor one residue at a time by utilizing a collection of nonsense suppressors that permit the insertion of 13 different amino acids in response to the amber (UAG) codon, as well as an additional amino acid in response to the UGA codon. We used this collection to suppress nonsense mutations at 141 positions in the lacI gene, which encodes the 360 amino acid long lac repressor, including 53 new nonsense mutations which we constructed by oligonucleotide-directed mutagenesis. This method has generated over 1600 single amino acid substitutions in the lac repressor. We have cataloged the effects of these replacements and have interpreted the results with the objective of gaining a better understanding of lac repressor structure, and protein structure in general. The DNA binding domain of the repressor, involving the amino-terminal 59 amino acids, is extremely sensitive to substitution, with 70% of the replacements resulting in the I- phenotype. However, the remaining 301 amino acid core of the repressor is strikingly tolerant of substitutions, with only 30% of the amino acids introduced causing the I- phenotype. This analysis reveals the location of sites in the protein involved in inducer binding, tighter binding to operator and thermal stability, and permits a virtual genetic image reconstruction of the lac repressor protein.     

Frameshifting in the expression of the Escherichia coli trpR gene

The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site-specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N-terminus of the +1 frame product and that of the known 0 frame product are identical but that their C-termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.     

DNA sequence and analysis of the bet genes encoding the osmoregulatory choline—glycine betaine pathway of Escherichia coli

The sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline-glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.8kDa); betT encoding a proton-motive-force-driven, high-affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of lambda placMu53 insertions, amino acid sequence similarity, or N-terminal amino acid sequencing. The bet genes are tightly spaced, with betT located upstream of, and transcribed divergently to, the tandemly linked betIBA genes.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.     

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP.     

Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies.

To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.     

Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator

The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.     

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.     

Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline resistance-gene-repressor

The nucleotide sequence of the pSC101-encoded tetracycline repressor gene (tetR) was confirmed. The deduced amino acid sequence is compared to that of other repressor proteins. To overproduce the repressor protein, tetR was placed under the control of bacteriophage lambda promoter pL. Tet repressor protein was purified to homogeneity and shown to bind specifically to two tet operators and also to tetracycline (Tc). The inducer function of Tc is demonstrated by the loss of the specific binding between the tet operator DNA and the Tet repressor-Tc complex.     

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.