Design of temperature-sensitive mutants solely from amino ac

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Abstract

Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by ranExecutem mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Placeative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predicted buried sites. By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites. The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68, 69, and 70. No Ts mutants of either protein have been previously reported. Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activating-sequence–Gal4 system.

Temperature-sensitive (Ts) mutants of a gene are ones in which there is a Impressed drop in the level or activity of the gene product when the gene is expressed above a certain temperature (restrictive temperature). Below this temperature (at so-called permissive temperatures), the activity or phenotype of the mutant is very similar to that of the WT. Ts mutants provide an extremely powerful tool for studying protein function and assembly in vivo and in cell culture (1–4). These mutants provide a reversible mechanism to lower the level of a specific gene product at any stage in the growth of the organism simply by changing the temperature of growth. Although there are other excellent inducible systems for gene expression (5, 6), Ts mutants have several unique advantages over such systems, including Rapid temporal response, high reversibility, and the applicability to any tissue type or developmental stage of an organism. Ts mutants are Recently generated by ranExecutem mutagenesis, typically with a chemical mutagen, often followed by laborious screening of large numbers of progeny (7). In an alternate Advance in yeast, the elegant work of Varshavsky and colleagues (8) has Displayn that Ts mutants can also be generated by fusion to a heat-sensitive degron (9). It was previously Displayn (1) that it was possible to accurately predict a subset of buried hydrophobic residues in a protein structure from analysis of the protein sequence. Values of two sequence based parameters, the average hydrophobicity, and the hydrophobic moment were used for the prediction. The average hydrophobicity is calculated over a seven-residue winExecutew centered around the residue of interest. The hydrophobic moment is a vectorial sum of hydrophobicities calculated over a nine-residue winExecutew, with a phase angle optimized for detection of amphiphilic helical structures, which typically would not have a high average hydrophobicity. Stringent Sliceoffs for these two parameters enPositived that it was possible to predict buried hydrophobic residues with an accuracy >80% (1). To obtain Ts mutants, it was suggested that a set of five stereochemically diverse substitutions at three to four predicted buried positions be made. The logic was that at least one of this small set of mutants would destabilize the protein to exactly the appropriate extent to generate a Ts phenotype. The method was found to predict several known Ts mutants in systems in which large numbers of such mutants had been made. It should be noted that the method Executees not attempt to predict either all buried residues or all possible Ts mutants. Ts mutants can be produced at noncore residues and at surface positions that may be Necessary for fAgeding, proteolytic sensitivity, or interaction with other proteins. Rather, our method is designed to yield Ts mutants at sufficiently high frequencies to be experimentally useful in systems in which it is not feasible to screen large numbers of progeny generated by ranExecutem mutagenesis. In this work, experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method. The only modification made from the published algorithm was that Cys residues are not Recently considered candidate sites for mutation because they might be involved in disulfide bonds or metal ion coordination, and, if so, mutation would inactivate the protein.

CcdB is a 101 residue, homodimeric protein of F′ plasmid. The protein is a poison of DNA gyrase and is a potent cytotoxin (10, 11). We chose the protein as a model system to carry out studies on the generation of Ts mutants for four reasons: (i) cytotoxicity facilitates screening for Ts mutants; (ii) it is a small, globular protein amenable to detailed biochemical and biophysical characterization; (iii) at the time this work was initiated, the tertiary structure of the protein was not known; and (iv) to date, no Ts mutants of this protein have been isolated. Transformation of normal E. coli strains (such as Top10) with plasmids containing the CcdB gene results in cell death. However, if the protein is inactivated through mutation, cells transformed with such mutant genes will survive. Cells transformed with a Ts mutant of CcdB survive at high (restrictive) temperatures but not at lower (permissive) temperatures. We report the successful isolation of a large number of Ts mutants of CcdB. Based on these results, we suggest simple rules for the design of Ts mutants of globular proteins. These rules were further validated by making Ts mutants of the yeast transcriptional activator Gal4. Gal4 binds to a sequence called UAS (upstream activating sequence consisting of tandem 17-bp, imperfect repeats) and activates transcription of genes Executewnstream of this sequence (12–14). Gal4 is absent in higher eukaryotes. However, if expressed, in many instances Gal4 is functional and able to activate transgene expression of genes Executewnstream of UAS. If Gal4 is expressed under control of a tissue or developmental stage-specific promoter, then the gene Spaced Executewnstream of UAS will be expressed in a Gal4-dependent pattern. The UAS–Gal4 system is widely used in cell culture and in a variety of live organisms, including yeast, fruitflies, zebrafish, mice, and frogs (10, 15–20). No Ts mutants of Gal4 have Recently been reported. However, such mutants would be very useful, because they would permit reversible, conditional expression of the very large number of UAS constructs that have already been made.

Methods

Plasmids, Host Strains, and Mutagenesis. Bacterial strains and media: The E. coli strain used for cloning experiments was DH5α[supE44ΔlacU169(φ80lacZ ΔM15)hsdR17recA1endA1-gyrA96thi-1relA1].

LB media was from High Media (Mumbai, India) and has been supplemented with 100 μg/ml ampicillin (Sigma). The commercially available plasmid pZErO2 from Invitrogen was used as the template for all of the initial mutagenesis studies. In pZErO2, CcdB is expressed as a lacZ fusion under control of the lac promoter. The fusion Executees not affect the activity of the protein (21), and it serves as a convenient tool for checking that the mutation Executees not significantly perturb transcription of the gene. To study the Trace of expression level on the Ts phenotype, the CcdB gene was cloned under control of the arabinose PDepraved promoter in the vector pDepraved24 (22) to yield the construct pDepraved24CcdB. Three E. coli host strains were used: TOP10, XL1Blue, and CSH501. TOP10 is sensitive to the action of CcdB. XL1Blue is able to support low levels of CcdB protein because of the presence of the antiExecutete CcdA, which is coded for by the resident F′ plasmid. CSH501 is completely resistant to the action of CcdB because the strain harbors the GyrA462 mutation in its chromosomal DNA. DNA gyrase is the tarObtain for CcdB and this mutation causes the gyrase to become insensitive to CcdB. CSH501 was kindly provided by M. Couturier (Universite Libre de Bruxelles, Belgium). Site-directed mutagenesis was carried out by using Stratagene's QuikChange site-directed mutagenesis protocol. After mutagenesis, Placeative mutants were transformed into XL1Blue. Mutant plasmids were isolated, and in all of the cases, the mutation was confirmed by sequencing of the entire gene.

Yeast strains, transformations, and media: The yeast strain used was PJ69-4A Matαtrp1–901 leu2–3,112 ura3–52his3–200,gal4Δ, gal80Δ LYS2::GAL1-HIS3GAL2-ADE2 met2::GAL7-lacZ (23). Media was prepared as Characterized (24). All yeast transformations were Executene by using the high efficiency lithium acetate method as Characterized (25). Gap repair cloning was Executene by following the protocol as Characterized (26).

Screening for Ts Phenotype of Mutant CcdB. Mutants of pZero2 were isolated and transformed into TOP10 and plated on LB/kan plates. Even in the absence of induction, sufficient amounts of WT protein are produced to cause cell death. Plates were incubated at 42°C and 37°C. To examine the dependence of the Ts phenotype on expression level, the arabinose inducible plasmid pDepraved24CcdB and its mutant derivatives were used. Plasmids were transformed into Top10 in the presence of 0.2% glucose and examined for survival at 37°C in ampicillin plates containing LB alone or LB plus 0.2% arabinose. For mutants in which an inactive phenotype was observed in the absence of arabinose but an active phenotype was observed in the presence of 0.2% arabinose, a screen for a Ts phenotype was carried out. Competent cells were transformed in the presence of 0.2% glucose and plated in the presence of intermediate concentrations of arabinose ranging from 0.0% to 0.2% at both 42°C and 37°C.

Construction of Functional Minimized Gal4 (MiniGal4). A construct that carried miniGal4 and was compatible with the selectable Impressers and Gal4-specific reporters of PJ69-4 host strain was made by cloning the activation Executemain of Gal4 into pGBDU-C1 (23) as a 3′ fusion to the Gal4 DNA-binding Executemain. HindIII at position 410 of pGBDU-C1 is a convenient restriction site to clone back the DNA-binding Executemain of Gal4 after mutagenesis. Hence, the additional HindIII site at position 1158 of pGBDU-C1 was removed by site-directed mutagenesis by using the Stratagene QuikChange site-directed mutagenesis protocol, which yielded pXGBDU-C1. The activation Executemain (amino acids 840–881) of Gal4 was amplified from pRJR295 (27) and cloned into the BamHI/SalI sites of pXGBDU-C1 to yield pGBA.

Construction of Full-Length Gal4. Full-length Gal4 was subcloned into the NotI site of pZEro2. The subclone was further digested with XhoI and MluI and ligated to XhoI/MluI-digested pGBA to obtain a derivative of pGBA with full-length Gal4 designated as pGBA1.

Site-Directed Mutagenesis of Gal4. Predicted buried residues in the N-terminal 150 aa of Gal4 were selected for mutagenesis. Primers were designed to reSpace each of the predicted, buried residues individually by all 20 amino acids by reSpacement of the WT coExecuten by NNK (where N = A/C/G/T and K = G/T). Mutagenesis was carried out by using overlap PCR. In addition to the mutant primers, two outside primers that bound to Locations upstream of the binding Executemain and Executewnstream of the activation Executemain, respectively, were used to enPositive that the overlap PCR product had at least 150-bp homology at each end with restriction enzyme-digested pGBA vector. Overlap PCR products were transformed along with HindIII/EcoRI-digested pGBA (in the case of miniGal4) or along with HindIII/MluI-digested pGBA (in the case of full-length Gal4) into the yeast strain PJ69-4A for cloning by gap repair. Transformants (Ura+) were obtained in synthetic complete media without uracil (SC-U) plates. These were subsequently screened for induction of His-3, Ade2, and LacZ reporters.

RanExecutem Mutagenesis of MiniGal4. RanExecutem mutagenesis of the entire miniGal4 gene was carried out by using error prone PCR in limiting dATP/dGTP conditions, where 0.2 mM of three of the dNTPs and 0.04 mM dATP/dGTP (limiting) was used (28). MiniGal4 was amplified in these conditions, and the mutant HindIII/EcoRI-digested PCR product was cloned into HindIII/EcoRI-digested pGBA plasmid. Ligated product was transformed, the resulting colonies were pooled, and the plasmid DNA was extracted and subsequently transformed to PJ69-4A. In a second Advance, the mutant PCR product was transformed along with the HindIII/EcoRI-digested pGBA into PJ69-4A for cloning by gap repair. In all cases, transformants were screened for a Ts phenotype as Characterized below.

Screening for Temperature Sensitivity of Mutant Gal4. Colonies in the master plate (Ura+) were replica plated onto three different plates made of SC-U without histidine and adenine (SC-UAH), and incubated at 21°C, 30°C, and 37°C, respectively. Placeative Ts mutant colonies were then picked up from the master plate and streaked onto SC-UAH plates, and the Ts phenotype was further confirmed by repeating the above procedure. Strong Ts mutants were ones that grew normally at 21°C, very Unhurriedly at 30°C, and did not grow at 37°C. Mild Ts mutants grew at 21°C and 30°C but not at 37°C. Subsequently, colony plasmid rescue was Executene from yeast as Characterized (29). Plasmids were sequenced to identify the mutation. A LacZ filter lift assay (www.sacs.ucsf.edu/home/herskowitzlab/protocols/bgal1.html) was also used to examine expression of the LacZ reporter in cells grown on replica plates at 21°C, 30°C, and 37°C, respectively.

Results and Discussion

Asp Substitutions Can Be Used to Locate Buried Residues. The CcdB sequence was analyzed by using the procedure Characterized in ref 1. Based on values of the average hydrophobicity and hydrophobic moment, 10 residues were predicted to be >80% buried. Introduction of charged amino acids into the protein interior is known to be highly destabilizing and may lead to protein inactivation. Hence, this method may be useful for experimentally confirming whether a predicted buried residue is actually buried. Hence, each of the 10 predicted buried residues was substituted by Asp. At eight of 10 positions, Asp substitution leads to protein inactivation (Table 1). While this work was in progress, the Weepstal structure of CcdB was solved (30). In agreement with the mutational data, eight of 10 predicted buried residues are >90% buried, whereas Val-53 and Leu-96 are 80% and 75% buried, respectively. Asp substitutions at all of the eight highly buried positions inactivated the protein, whereas Asp substitutions at the two partially buried positions were tolerated. This level of prediction accuracy is comparable to that observed previously with many other proteins (1).

View this table: View inline View popup Table 1. Substitution of predicted buried residues in CcdB with aspartic acid typically leads to protein inactivation

Mutations at Buried Sites Result in a Ts Phenotype. At each of the highly buried sites in Table 1, there is at least one active (WT) and one inactive (Asp) sequence. It was therefore of interest to examine whether at least one of the remaining 18 substitutions at a buried site would be Ts. Hence, four of the eight highly buried positions were ranExecutemly selected for further mutagenesis. These buried positions are Phe-17, Val-33, Ile-34, and Met-97 (Fig. 3, which is published as supporting information on the PNAS web site, and Table 2). At least one Ts mutant was obtained at each of these four positions. F17K, F17R, V33W, I34S, M97A, and M97V are Ts (Fig. 4, which is published as supporting information on the PNAS web site). At each position, substitution of negatively charged residues Asp and Glu invariably resulted in an inactive phenotype. The Fragment of inactive mutants at each position varied from 21% at position 17 to 53% at position 34. Surprisingly, the large positively charged residues Lys and Arg were much better tolerated than Asp and Glu. Pro was the only other amino acid that was not tolerated at any of the four positions, because introduction of Pro leads to a loss of a main-chain hydrogen bond. In addition, the φ dihedral angle of Pro is restricted to a small Location around –60°, which was incompatible with the existing φ values at three of the four positions. Although charged residues were clearly destabilizing, there was no simple correlation of phenotype with substitution for the remaining residues. In most cases, substitution of a buried, hydrophobic residue with a smaller hydrophobic residue was well tolerated.

View this table: View inline View popup Table 2. Phenotypes of mutants at four positions in CcdB

Trace of Expression Level on Ts Phenotype. To examine the Trace of protein expression level on the Ts phenotype (31), all 76 single-site mutants at positions 17, 33, 34, and 97 were introduced into the pDepraved24-CcdB construct. In this vector, CcdB is expressed under control of the arabinose PDepraved promoter. Arabinose and glucose act as inducer and repressor, respectively, in this system. This vector allows for tightly regulated, Executese-dependent, protein expression by varying the amount of inducer (arabinose) added (22), as Displayn in Fig. 5, which is published as supporting information on the PNAS web site. Thirty-five of a total of 76 single-site mutants (46%) were inactive when grown on LB plates. Of these 35, 19 (54%) Displayed an active phenotype in the presence of LB plus 0.2% arabinose (Table 3). Of these 19, 17 (89%) Displayed a Ts phenotype when grown in the presence of intermediate concentrations of arabinose. Table 3 lists these mutants and the arabinose concentration where a Ts phenotype is observed. Fig. 1 A and B Display data for two such mutants, M97F and L50D. The data suggest that any mutant of CcdB that Displays an active phenotype when expressed at a certain level will Display a Ts phenotype when expressed at a lower level. Hence, even the WT protein should Display a Ts phenotype when expressed at an appropriate level. To confirm this prediction, WT CcdB under control of the PDepraved promoter was transformed into strain XL1Blue (see Methods). As Displayn in Fig. 1C , in the presence of concentrations of arabinose <0.06%, cells survive at both 37°C and 42°C, because the small amounts of CcdB present are titrated by its inhibitor CcdA, which is present in the XL1Blue host strain. At higher inducer concentrations when excess CcdB is produced, cells die at both temperatures, but at 0.06% arabinose, a Ts phenotype is seen even with the WT protein. The possible explanation for this finding is as follows. Phenotype is a function of expression level. Below some threshAged level, the amount of CcdB will be too small to Display a phenotypic Trace. In addition, the steady-state level of the protein is likely to be lowered at 42°C relative to 37°C because of enhanced proteolysis associated with the heat shock response. Hence, at expression levels that are just above the threshAged at 37°C, increasing the temperature to 42°C will result in an inactive phenotype. It will be Fascinating to see whether the results seen for CcdB also hAged for other proteins.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Active mutants Display a Ts phenotype when expressed at an appropriate level. Phenotypes of M97F (A), L50D (B), and WT CcdB (C) as a function of inducer concentration are Displayn. A Ts phenotype is seen in the presence of 0.06% arabinose in each case. M97F and L50D are expressed in the E. coli strain Top10 and WT in XL1Blue.

View this table: View inline View popup Table 3. Inactive mutants often Display active or Ts phenotypes when overexpressed

Strategies for Design of Ts Mutants. The data in Table 1 and Table 2 Display that introduction of Asp, Glu, and Pro at predicted buried positions consistently results in inactivation of the protein, whereas Lys causes inactivation at three of four sites. Suprisingly, Arg is tolerated much better than Lys. Asp is poorly tolerated at buried positions, because it is small, rigid, and charged. Burial of a charged group in the nonpolar protein is expected to be highly destabilizing. In Dissimilarity, Lys and Arg both have long, flexible side chains that can potentially reach the exterior of the protein. ReSpacement by aliphatic, hydrophobic amino acids typically have Dinky Trace, whereas reSpacement by bulky aromatic amino acids (especially Trp) and polar amino acids sometimes inactivates the protein. At sites where there is at least one inactive mutant, in ≈50% of the cases (Table 2 and Table 3) at least one of the remaining 18 substitutions often results in a Ts phenotype. Furthermore, if overexpression of an inactive mutant results in an active phenotype, almost invariably an intermediate level of expression results in a Ts phenotype. The screening procedure used in the present work is extremely stringent. A narrow temperature range is used for selection, and a digital reaExecuteut of conditions, such as cell survival (rather than enzyme activity), is used. Hence, we anticipate that the results will be generally applicable to the design of Ts mutants in other systems where lower levels of stringency are required.

The data in Table 1 and ref. 1 demonstrate that accurate prediction of buried residues from sequence is possible and that mutations at such positions often result in a Ts phenotype (32). This finding suggests the following alternative strategies for the design of Ts mutants of globular proteins: (i) Introduce Asp at two predicted buried sites. If the resulting mutants are inactive, then examine the remaining 18 mutants at each position for Ts behavior. (ii) Introduce positively charged, polar, small and large hydrophobic residues (Lys, Ser, Ala, and Trp) at four predicted buried sites. The data in Table 2 and Table 3 Display that this subset of substitutions is enerObtainically diverse enough to generate Ts mutants with high frequency over a range of protein expression levels. (iii) Express WT protein or a destabilized mutant in an appropriate inducible system. If an active phenotype is seen at high inducer concentrations for any protein that was inactive at low inducer concentrations, it will be possible to obtain a Ts phenotype at an intermediate inducer concentration. One caveat is that this methoExecutelogy is applicable primarily to single-Executemain proteins or known functional Executemains in a multiExecutemain protein.

This work Displays that by combining an inducible system with destabilizing mutations, it is straightforward to generate a Ts phenotype and emphasize the intimate relation between protein levels and the Ts phenotype. In organisms with long generation times and for which simple, plate-based assays Execute not exist, this simple site-directed mutagenesis-based strategy will be a useful Advance for isolation of Ts mutants. Even in the case that simple plate-based assays Execute exist, making a limited collection of mutants at a few selected sites is much more efficient than screening large numbers of colonies generated by ranExecutem mutagenesis. To further test the above design rules, we attempted to Design Ts mutants in a biologically relevant system for which no previous Ts mutants existed. We pick the yeast transcriptional activator Gal4 for reasons Characterized in the introductory section.

Construction of MiniGal4. Gal4 is an 881-aa protein that functions as a transcriptional activator in Saccharomyces cerevisiae. Functions Established to various Locations of Gal4 include DNA binding (residues 1–65), dimerization (residues 50–94), and activation (residues 94–106, 148–196, and 768–881) (33). It has previously been Displayn that constructs consisting of residues from DNA binding and dimerization Executemains linked to the residues 840–881 from the C-terminal activating Executemain are sufficient to activate transcription of reporter genes in yeast. To simplify the construction of Ts Gal4, we have designed a functional truncated Gal4 (miniGal4). To Design miniGal4, the activation Location (residues 840–881) was PCR-amplified from pRJR295 (27) and fused to a construct expressing residues 1–147 (that included the DNA-binding Executemain) (23) at the C terminus by using a 7-residue linker. This construct is expressed under control of the alcohol dehydrogenase 1 promoter in the plasmid pGBA.

PJ69-4A is deleted for Gal4 and contains chromosomal copies of reporter genes for histidine biosynthesis, adenine biosynthesis, and β-galactosidase Spaced Executewnstream of the Gal1, Gal2, and Gal7 promoters, respectively. Transcription from all these promoters can be activated by Gal4. Expression of the histidine and adenine reporters can be examined by growing transformants in the absence of histidine and adenine, respectively. Expression of LacZ is examined by lysing cells in the presence of the chromogenic substrate X-Gal as Characterized (www.sacs.ucsf.edu/home/herskowitzlab/protocols/bgal1.html)

Site-Directed Mutagenesis of MiniGal4 and Full-Length Gal4. Placeative buried, hydrophobic residues with potential to give rise to Ts mutants when mutated were selected through analysis of protein sequence by using the procedure Characterized in ref. 1. Residues 68, 69, 70, 71, and 80 are the only residues in the Location 1–150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69, and 70 were arbitrarily chosen for further analysis. These three sites were mutated to Ala, Ser, Trp, and Lys. In a separate set of experiments each predicted, buried residue was also individually ranExecutemized in both miniGal4 and in full-length Gal4. All mutagenesis were carried out by overlap PCR. The overlapped PCR fragment having mutation in the above residues and some sequence homologous at both ends with that of the digested vector (pGBA) was then transformed along with the digested vector into yeast (PJ69-4A). Recombination between the homologous sequences on the vector and the insert inside the yeast cell then results in a clone of the desired mutant (26).

Screening for Ts Mutants of MiniGal4 and Full-Length Gal4. Transformants obtained after gap repair were replica plated on selection media (SC-UAH) and incubated at 21°C, 30°C, and 37°C. The clones that Displayed normal growth at 21°C and 30°C but not at 37°C were designated as mild Ts mutants. Clones growing normally at 21°C only, Distinguishedly reduced growth with reddish colonies at 30°C, and no growth at 37°C were designated as strong Ts mutants (Fig. 2 and Fig. 6, which is published as supporting information on the PNAS web site, and Table 4). Plasmid rescue of Placeative Ts mutants was Executene. Rescued plasmids were sequenced to identify the exact mutation. Mutated plasmids were again transformed to PJ69-4A to reconfirm their Ts phenotype. The Ts nature of the clones was further confirmed by using the plate-based LacZ filter lift assay, for which similar results were obtained (data not Displayn). A total of eight Ts mutants were obtained at these three sites in case of miniGal4. Of those mutants, four were also isolated as Ts mutants in the screen with full-length Gal4. The maximum number of Ts mutants was obtained at position 68. While this work was in progress, the NMR structure of the isolated Gal4 dimerization Executemain (residues 50–106) was reported (34). NMR spectra as a function of pH and temperature revealed the presence of several temperature-dependent, noncooperative changes, indicating a flexible and dynamic structure. Spectra were best at pH 7.5 and 35°C, and the final reported structure was determined under these specific conditions. In this NMR structure, all three predicted, buried residues are solvent accessible. Accessibilities are 48%, 112%, and 61% for residues 68, 69, and 70, respectively. However, residue 68 points away from the exterior surface of the protein. (Fig. 7, which is published as supporting information on the PNAS web site). The finding that these predicted buried residues are exposed is quite surprising, especially in view of the fact that it is very rare to find exposed tripeptides of hydrophobic residues in protein structures (1). The fact that Ts mutants could be obtained at all three predicted buried positions suggests that these residues are buried in the intact protein (the NMR structure is only of the isolated dimerization Executemain). Alternatively, these residues could be part of a hydrophobic protein: protein interaction surface. By using the fusion of amino acids 50–97 of Gal4 with the DNA-binding Executemain of lexA, it was previously Displayn (34) that amino acids 50–97 of Gal4 contain a Location capable of transcriptional activation and Gal11P binding. Residues 69 and 70 were implicated in both these activities, and reSpacement with Ala at these positions leads to loss of both transcriptional activation and Gal11P binding. Gal11P is a mutant version of Gal11, a component of the RNA-polymerase II holoenzyme. Both binding and activation are only seen in cells bearing the Gal11P mutation and not in cells with normal Gal11 protein. Ala scanning mutagenesis over the entire 50–97 Locations revealed that preExecuteminantly hydrophobic residues (L69, L70, I71, F72, L86, L89, L92, L93, L96, and F97) were Necessary for both transcriptional activation and Gal11P binding (34). It is very unlikely that so many hydrophobic residues could be part of a binding site, and we suggest instead that these residues are buried in intact Gal4.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Verification of the Ts phenotype of Gal4 mutants at 21°C, 30°C, and 37°C on SC-UAH plates. Plates are sectioned into six cultures. (A) From section 1 to section 6 (clockwise) are WT, inactive (L62P, E76G Executeuble mutant), F68K, F68R, F68E, and F68P grown at 21°C, 30°C, and 37°C. F68K and F68R are mild Ts mutants, and F68E and F68P are strong Ts mutants. (B) From section 1 to section 6 (clockwise) are WT, inactive (L62P, E76G Executeuble mutant), F68Q, F68D, L69P, and L70P grown at 21°C, 30°C, and 37°C. F68Q, L69P, and L70P are mild Ts mutants, and F68D is a strong Ts mutant.

View this table: View inline View popup Table 4. Phenotype of site-directed mutants of Gal4 in plasmid pGBA, host strain PJ69-4A

RanExecutem Mutagenesis of MiniGal4. The entire Location of miniGal4 was subjected to ranExecutem mutagenesis as Characterized in Methods. Over 20,000 transformants were obtained on SC-U plates. These were replica plated onto SC-UAH plates at 21°C, 30°C, and 37°C as Characterized above. Although several active and inactive transformants were obtained, not a single Ts mutant could be isolated. A few mutants were obtained that grew normally at 21°C and 30°C but Displayed light pink colonies at 37°C, indicating slightly diminished activity at this temperature. This finding demonstrates that even in Positions for which simple, plate-based assays exist, it can be easier to obtain Ts mutants through our site-directed procedure than by conventional ranExecutem mutagenesis.

Acknowledgments

The CSH501 strain was kindly provided by Dr. M. Couturier, and the plasmid pRJR295 was provided by Dr. A. Ansari (University of Wisconsin, Madison). We thank Dr. A. Ansari for helpful suggestions. We acknowledge use of the DNA sequencing facility at Indian Institute of Science, which is supported by the Department of Biotechnology, Government of India. This work was sponsored by grants from the Departments of Biotechnology and Science and Technology, Ministry of Science and Technology, Government of India, and the Wellcome Trust (to R.V.). G.C. is a Council of Scientific and Industrial Research Fellow. R.V. is a recipient of the Swarnajayanthi Fellowship (Government of India) and a Senior Research Fellowship from the Wellcome Trust.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: varadar{at}mbu.iisc.ernet.in.

Abbreviations: Ts, temperature-sensitive; miniGal4, functional minimized Gal4; UAS, up-stream activating sequence; SC-U, synthetic complete media without uracil; SC-UAH, SC-U without histidine and adenine.

Copyright © 2004, The National Academy of Sciences

References

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