Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Second-Site Revertants of Escherichia Coli Trp Repressor Mutants

Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli. These mutants were altered throughout the gene. The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the liklihood that the revertants obtained would have second-site changes. Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes. These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan. Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors.     

Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites.

A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altered specificities. The library members contain one wild-type DBD and one mutant domain with randomized amino acids in the DNA-contacting region. Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and NNNN by the mutant domain. In principle, any sequence can stand for NNNN and serve as a selection target. Here an in vivo library screening method was used to isolate mutant sc repressors that interact with an asymmetric operator containing the TTAA target. Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT. Specificity studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target. Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT.     

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.     

Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.     

Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139-4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene. Six of the I-d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303-307], the His-18 repressor did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.     

Negative Dominant Mutations of the uidR Gene in ESCHERICHIA COLI: Genetic Proof for a Cooperative Regulation of uidA Expression

The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.     

The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo.

The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo.     

Design of temperature-sensitive penicillinase repressors by replacement of Pro in predicted beta-turn structures.

Pro residues in predicted beta-turn structures were substituted with other amino acids to obtain temperature-sensitive penicillinase repressors (PenI). A mutant repressor (P70L; Pro-70 is substituted with Leu) was inactive at 48 degrees C and penP gene expression was derepressed (1,200 U/OD660 [optical density at 660 nm] ), although the mutant was still active at 30 degrees C (27 U). The heat induction ratio (penicillinase activity at 48 degrees C compared with that at 30 degrees C) of the mutant was 98 times higher than that of the wild type (i.e., 44 versus 0.45). This result indicated that the side chain of the Leu residue in P70L destroyed the proper folding of the repressor protein at the elevated temperature, whereas the Pro residue of the wild-type repressor stabilized this predicted beta-turn structure even at 48 degrees C. When the Pro residue was replaced by amino acid residues with smaller side chains (i.e., Gly and Ala), these mutant repressors were less temperature sensitive than P70L. These data suggest that the presence of the Pro residue in the beta-turn structure could be one of the key factors in stabilizing protein structure at elevated temperatures.     

DNA Sequence Analysis of Artificially Evolved Ebg Enzyme and Ebg Repressor Genes

The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.     

Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.     

Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

INTRODUCTIONThe transition from vegetative growth to reproductionis one of the most important developmental events in flow-ering plants since it is related to the competence and sur-vivability of a particular species living in a particularenvironment. The…     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.