1 Department of Biochemistry and 2 Department of Biophysics, Bose Institute, Acaharya J.C. Bose Birth Centenary Building, P-1/12, CIT Scheme VII M, Calcutta 700 054, India
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: bacteriophage /lambda repressor/structure of
repressor/temperature-sensitive mutant repressors of
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The repressor of bacteriophage , the product of its cI gene, negatively regulates the expression of the genes from the two early promoters, PL and PR, by specific interaction with two operators, OL and OR, respectively (Ptashne et al., 1980
; Ptashne, 1992
). The functional form of the repressor is a dimer of two identical subunits. The repressor binds to three operator sites, OR1, OR2 and OR3, that constitute the operator OR, in order of decreasing affinity with alternate pair-wise cooperativity. The selectivity and cooperativity are essential for proper functioning of the switch. The cooperativity is a consequence of proteinprotein interaction between two repressor dimers bound to two operator sites.
The repressor monomer has 236 amino acid residues and two functional domains: the N-terminal domain spanning from 1 to 92 and the C-terminal domain from 132 to 236 residues. The N-terminal domain is responsible for specific operator binding and contact with RNA polymerase for activation of PRM promoter, while the C-terminal domain is responsible for dimer formation and higher order proteinprotein contacts. Recent studies suggest that a communication occurs between the two domains of
repressor through conformational changes, and the nature of the proteinprotein interaction is significantly modulated following binding of the repressor with the operator sites (Saha et al., 1992
; Bandyopadhyay et al., 1995
). The amino acid sequence of the repressor has been determined by both DNA sequencing of the cI gene (Sanger et al., 1982
) and protein sequencing of the purified repressor (Sauer and Anderegg, 1978
). The intact
repressor has not been crystallized, but the crystal structures of the N-terminal domain (Pabo and Lewis, 1982
) and its complex with the operator DNA (Jordan and Pabo, 1988
) have been solved. Normal affinity of the wild-type repressor for operators could be altered by mutations. Thus, both low- and high-affinity mutations in the cI gene in phage (Kaiser, 1957
; Sussman and Jacob, 1962
; Lieb, 1966
; Nag et al., 1984
) and the latter type of mutations in plasmid-borne cI gene (Nelson and Sauer, 1985
) have been isolated. Recently, several cI mutants having mutations in the C-terminal domain have also been isolated which are defective in monomerdimer equilibrium and cooperative binding; some of these non-cooperative mutants have been studied in detail using the tools of structural biology (Burz and Ackers, 1994
; Burz et al., 1994
). This has led to the identification of several residues that are important in various proteinprotein interactions.
There are several ts mutations in the cI gene of which are distributed in the two functional domains (Lieb, 1966
). However, no ts mutation mapping in the connecting hinge region (93131) has been reported. It has been reported that all of these ts-mutant repressors lose DNA-binding activity at 42°C in cell-free extracts (Mandal and Lieb, 1976
). In vivo, certain ts mutations in the N-terminal domain complement certain others in the C-terminal domain in trans (Lieb, 1966
, 1976
). However, when any two of these mutations are present in cis in the same cI gene, the resulting repressor is non-functional even at 30°C (Nag et al., 1982
). The existence of many of these cIts mutants of
with such interesting properties led us to initiate the characterization and detailed structural studies of the ts-mutant repressors. In this work, we determined the changes of amino acids induced by seven different ts mutations and their exact positions in the repressor protein by sequencing DNA of the relevant mutant cI genes. As the lysogen of
cIts2 mutant was shown to be the most temperature sensitive and most susceptible to UV induction among the known
cIts mutants (Lieb, 1966
), and in this study we found that the mutant CIts2-protein carries a charge-reversal K224E substitution, a detailed structural investigation on this mutant repressor was also carried out.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acrylamide, DTNB, ß-mercaptoethanol, IPTG, PMSF, bovine serum albumin (BSA), DTT, calf thymus DNA, glycerol, DNase, RNase, QAE-Sephadex and polyethyleneimine were purchased from Sigma Chemical (St Louis, MO). Restriction enzymes were purchased from GENEI (Bangalore, India). Bactotryptone, bactoagar and yeast extract were obtained from Difco Laboratories (Detroit, MI) and hydroxyapatite from Bio-Rad (USA). The DNA sequencing kit Sequenase version 2.0 was bought from Amersham. Oligo primers for DNA sequencing were synthesized in an automated DNA synthesizer (Applied Biosystems, Model 380A). [32P-]dATP and [3H]thymidine were obtained from Bhaba Atomic Research Center (Mumbai, India). Millipore filters (HAWP, HA 0.45 µm, 25 mm) were purchased from Millipore India (Bangalore, India). All other chemicals and reagents were of analytical grade and were purchased from local suppliers.
Bacteria, bacteriophages and plasmids
Escherichia coli RR1 [supE44 hsdS20 ara14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl1] and DH5 (supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) were obtained from S.Adhya (National Institutes of Health, Bethesda, MD). E.coli 594 (F galK galT lac strr su) and Y-mel (suIII+) and bacteriophage strains,
cI,
cItsU50,
cItsU9,
cItsU46,
cIts1,
cItsU51,
cItsI-22 and
cIts2 from M.Lieb. The plasmid pEA305 (Amann et al., 1983
) was a gift from M.Ptashne. pNM1 (Das and Mandal, 1986
) and pBR322 were used from laboratory stocks. Other plasmids were constructed during this work.
Bacterial growth
E.coli was routinely grown in tryptone broth (TB) (1% bactotryptone and 0.5% NaCl) or LuriaBertani broth (LB) (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.1) with shaking at the desired temperature. The growth was monitored by measuring the optical density (OD) at 590 nm. [3H] DNA was prepared as described by Mandal et al. (1974).
Recombinant DNA methods
Transformation of plasmid DNA in E.coli was done by the CaCl2 method as described by Sambrook et al. (1989). The strategies of cloning of the mutant cIts genes of are described in Figure 1
.
|
The mutant cIts genes from seven different cIts mutant phages,
cItsU50,
cItsU9,
cItsU46,
cIts1,
cItsU51,
cItsI-22 and
cIts2 were cloned in pBR322 (see Figure 1
for details) to obtain the plasmids pMJU50, pMJU9, pMJU46, pMJ1, pMJU51, pMJI-22 and pMJ2, respectively.
DNA sequencing
The nucleotide sequence of the complete DNA molecule is known (Sanger et al., 1982
). Hence four suitable 20-mer primers were designed such that the whole of the cI gene in a plasmid could be sequenced using those primers. The above mutant cI genes in plasmid were sequenced by the double-stranded DNA-sequencing method of Sanger et al. (1977) using synthetic oligo primers and the plasmid DNAs carrying the mutant cIts genes as templates and the Sequenase kit (version 2.0). Each mutation was sequenced at least three times from three independent sequencing reactions using the respective plasmid clones isolated from different colonies.
Subcloning of cIts2-mutant gene in an expression vector
The wild-type cI gene of is expressed under the control of tac promoter in a plasmid pEA305 (Amann et al., 1983
) to a very high level. We first prepared a cassette plasmid pMS1 which could be used for subcloning most of the above cIts mutations for high expression under the control of tac promoter (see Figure 1
). The cIts2 mutation was subcloned in pMS1 to give pMSJ2. The presence of ts2 mutation in the cI gene tagged to the tac promoter in pMSJ2 was confirmed by restriction analysis and DNA sequencing: the high level of expression of the repressor was confirmed by SDSPAGE analysis of the extract of IPTG-induced cells carrying pMSJ2 (data not shown).
Isolation of repressors
Wild-type repressor was isolated from E.coli RR1 carrying pEA305 plasmid and the ts2 repressor from E.coli RR1 bearing the plasmid pMSJ2 (see Figure 1). The cells containing the above plasmids were separately grown to an OD590 of 0.6, IPTG was added to a final concentration of 1 mM and the plasmids were grown further for 2 h at the same temperature. The cells were then harvested and washed and the pelleted cells were stored at 20°C. The wild-type repressor was purified following exactly the procedure described by Saha et al. (1992). The ts2-mutant repressor was purified basically by the above procedure with the following modifications. After centrifugation of the sonicated extract at 12 000 g, the supernatant was subjected to ammonium sulfate fractionation. The precipitate obtained at 6080% saturation was dissolved in sonication buffer [10 mM TrisHCl, pH 8.0, 0.1 mM EDTA, 2 mM CaCl2, 0.1 mM ß-mercaptoethanol and 5% (v/v) glycerol] containing 25 mM KCl. This was then subjected to QAE-Sephadex column (instead of CM-Sephadex column) chromatography followed by hydroxyapatite column chromatography. The purified repressors were stored in the presence of 50% glycerol at 20°C. The repressor was assayed by the DNA filter-binding method of Riggs et al. (1968) and the protein concentration was determined from the relation A1%280 = 11.3.
Determination of sulfhydryl reactivity
The sulfhydryl reactivity was determined by the procedure described by Banik et al. (1992). This was done by titrating protein with DTNB under various conditions when 1 mol of 2-nitro-5-mercaptobenzoate was liberated per mole of exposed sulfhydryl (SH) group in the protein. This liberated mercaptobenzoate was quantitated from its absorption at 412 nm using the relation = 1.36 x 104 l/mol. cm at pH 8.0 (Ellman, 1959
).
Fluorescence methods
For all spectroscopic studies, the repressors were dialyzed against 0.1 M potassium phosphate buffer, pH 8.0. All fluorescence spectra were measured in a Hitachi F 3000 spectrofluorimeter with a computer for spectral addition and subtraction. For tryptophan fluorescence, the excitation and emission wavelengths were kept at 295 and 340 nm, respectively. All fluorescence values were corrected for volume changes, inner filter effects and blank values according to Bandyopadhyay et al. (1995). The excitation and emission bandpass were 5 nm unless mentioned otherwise. Acrylamide quenching of tryptophan fluorescence was studied as described by Bandyopadhyay et al. (1995).
Circular dichroism
Far-UV-circular dichroism (CD) spectra were measured in a Jasco J600 spectropolarimeter using a 10 mm pathlength cuvette at the required temperature controlled at ±1°C. The scan speed was 50 nm/min and 10 scans were signal-averaged to increase the signal-to-noise ratio.
Size-exclusion HPLC
Size-exclusion HPLC was performed with a Waters HPLC system using a Protein Pak TM 300 SW column (7.5 x 300 mm) having a fractionation range of molecular weight 10 000400 000 Da native globular proteins. The column was pre-equilibrated and eluted with 0.1 M phosphate buffer, pH 8.0, and a 0.4 ml/min flow rate was used throughout. Carbonic anhydrase, bovine serum albumin, yeast alcohol dehydrogenase, ß-amylase and apoferritin were used as molecular weight markers.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Seven cIts mutations, cItsU50, cItsU9, cItsU46, cIts1, cItsU51, cItsI-22 and cIts2 were sequenced using the plasmids pMJU50, pMJU9, pMJU46, pMJ1, pMJU51, pMJI-22 and pMJ2, respectively, as described in Materials and methods. The results are summarized in Table I. Of the four ts mutations in the DNA-binding domain, U50 and U46 have changes at the 21st (Ile to Ser) and 62nd (Ala to Thr) positions in the
-helices 1 and 4, respectively; U9 has replaced Gly by Ser at the 53rd position just outside the helix 3 on the C-terminal side, while ts1 has a change at the 73rd position (Val to Ala) in between the helices 4 and 5. Of the three ts mutations in the oligomerizing domain, cItsU51 carries two changes, one at the 141st (Phe to Ile) and the other at the 153rd (Pro to Leu) position. The cItsI-22 mutation has replaced Asn by Thr at position 207. In ts2 repressor, a charge reversal has occurred at position 224 (Lys to Glu), which is located very close to the C terminus.
|
The ts2 mutant repressor is very heat labile and highly sensitive to UV induction in a lysogen (Lieb, 1966). Its DNA-binding activity in cell-free extract prepared from a
cIts2 lysogen could not be demonstrated even at 2030°C (Mandal and Lieb, 1976
). Association of all these interesting properties with the CIts2 repressor led us to concentrate further on the structure and function studies of this mutant gene-regulatory protein.
The CIts2 repressor was purified by the procedure of Saha et al. (1992) with a slight modification as described in Materials and methods. It was observed that this mutant protein did not bind to CM-Sephadex to which the wild-type repressor could bind. On the other hand, this mutant repressor bound very tightly to QAE-Sephadex, to which the wild-type repressor bound very weakly. The purified ts2 repressor obtained from the hydroxyapatite column gave a single band corresponding to a monomeric molecular weight of 26 kDa on SDSPAGE gel (data not shown).
Operator-binding activity of ts2 repressor at permissive and non-permissive temperatures
Figure 2 shows the binding of ts2 repressor to whole
DNA containing wild-type OL (OL1, OL2 and OL3) and OR (OR1, OR2 and OR3) at 20°C. As only the dimer form of
repressor binds to operator DNA, the nature of the sigmoidicity in the curve showing the binding of repressor to the operator DNA as a function of the protein concentrations may be taken as a qualitative measure of monomermonomer association of the protein to form dimers and their subsequent cooperative interaction. It is clear from the data in Figure 2A
that the wild-type repressor shows sigmoidicity in the 02 nM range of protein concentrations, while the ts2-mutant repressor shows much less prominent sigmoidicity in the 26 nM concentration range. This possibly suggests a much weaker association of the ts2 monomer to form dimer, especially at low concentrations of the protein, and/or a defect in the cooperative interaction of the dimers. Even at higher concentrations of protein (1620 nM) where the plateau in binding reached, the mutant protein showed about 57% binding compared with the wild-type repressor under identical conditions. This indicates that even at higher concentrations of the protein, either the mutant dimer formation is not as efficient as that of the wild-type repressor and/or the binding of mutant repressor may not be stabilized well owing to weaker cooperative (tetrameric) interaction of the adjacently bound repressor dimers. The fact that a
cIts2 lysogen is relatively more susceptible to UV induction than a wild-type
lysogen supports the above views.
|
Structure of the C-terminal domain of ts2 repressor is different from that of the wild-type repressor
Clearly, the ts2-mutant repressor has a weak DNA-binding activity compared with the wild-type, even at permissive temperatures. Thus, a study of the structure of this mutant protein may shed much light on the state of the C-terminal domain. Figure 3 shows the CD spectra of the wild-type and ts2 repressors at 25°C. The spectrum of the mutant repressor is clearly different from that of the wild-type, with a significant loss of intensity at ~220 nm and an enhancement at ~206208 nm. If we assume that the secondary structure of the N-terminal domain is unchanged (the ts2 mutation is located at the C-terminal end), it is likely that the secondary structure of the C-terminal domain is significantly changed since the CD spectrum of the whole repressor is dominated by the spectrum of the helix-dominated N-terminal domain. This suggests that even at permissive temperatures, the mutant protein may be partially denatured.
|
|
Tryptophan fluorescence of a protein is often used as a sensitive probe for studying its structure. The repressor has three Trp residues, two of which are situated within the C-terminal domain and the third in the hinge region bordering the C-terminal domain. Acrylamide quenching of these Trp residues has been used to resolve conformational and structural aspects of the C-terminal domain of
repressor (Bandyopadhyay et al., 1995
). It has been shown that at 25°C in the presence of low concentrations of acrylamide, the fluorescence of wild-type repressor at 0.5 µM concentrations is quenchable to the extent of 40% (Ksv = 45 M1) and that at 10 µM to only about 25% (Ksv = 38 M1) (Bandyopadhyay et al., 1995
). The Lehrer plot of acrylamide quenching of the ts2-mutant protein at 25°C using 0.5 and 10 µM concentrations is shown in Figure 4
. The plots are similar at these two concentrations of the protein with only a modest difference in the initial part of the quenching. At 10 µM concentration of ts2 repressor, the plot is approximately linear and cuts the y-axis at ~1 with a Ksv of 10 M1 (Figure 4
). This suggest a much enhanced exposure of the two buried Trp residues at 25°C [Ksv of the buried tryptophans in the wild-type protein is ~3 M1 or lower (Bandyopadhyay et al., 1995
)]. At 42°C, the Lehrer plot for ts2 repressor at 0.5 µM cuts the y-axis at ~1 with a Ksv of 29 M1 (Figure 5A and B
). This is in contrast to that of the wild-type protein, which shows a biphasic behavior and cuts the y-axis at ~2. The two phases have Ksv of 29 and 9 M1, respectively (summarized in Table III
). This again suggests that the Trp residues which are buried in the wild-type protein are completely exposed in the ts2-mutant protein at non-permissive temperatures.
|
|
|
Lambda repressor is known to tetramerize in solution (Brack and Pirrotta, 1975; Banik et al., 1993
). In a previous study, it has been shown that the tetramerization of repressor is accompanied by a dramatic effect on the Trp230 residue resulting in an overall shift of emission maxima of 2 nm in buffers containing 0.5 M acrylamide (Bandyopadhyay et al., 1995
). At comparable protein concentrations at 25°C, the tryptophan fluorescence of ts2 repressor was found to be red shifted (data not shown). Figure 6
shows the emission maximum of tryptophan fluorescence of ts2 and wild-type repressors in 0.5 M acrylamide as a function of protein concentration at 25°C. As noted previously, the wild-type repressor showed a blue shift of ~2 nm when the protein concentration was changed from 0.2 to 10 µM. The ts2 repressor, however, showed a blue shift of only a few tenths of a nanometer in the same protein concentration range. Clearly, the change of W230 environment, which is characteristic of the wild-type protein association, is absent in the ts2 repressor. In order to determine whether this lack of environment change in the latter repressor was due to the lack of tetramer formation, we performed size-exclusion HPLC at 25°C. The ts2 protein eluted as a dimer at all loading concentrations in the range 150 µM, whereas under identical conditions, the wild-type repressor showed a significant shift towards higher molecular weight species (data not shown). This suggests that the ts2 protein is defective in tetramer formation. Hence it appears that the lack of emission maxima shift in the mutant protein is probably due to the lack of tetramer formation in this concentration range. Taken together with the operator-binding data in Figure 2A
, this indicates that at permissive temperatures, the cooperativity of ts2 repressor may be weaker than that of the wild-type.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several ts mutations in the cI gene of originally isolated in phage show a nearly even distribution in the N- and C-terminal domains of the repressor (Lieb, 1976
). In this work, the exact positions and the amino acid changes due to seven such cIts mutations were determined by DNA sequencing (Table I
). It appears that all seven cIts mutations show base changes at either the first (5') or second (middle) position but not at the third (3') position of the codon; this is possibly due to the third base flexibility in the codon dictionary. In this group of cIts mutants, no mutation was found in the hinge region between residues 92 and 132. The amino acid changes caused by these mutations appear to be random with respect to their different properties such as the polarity, charge and size of the side-chain. The mutations tsU50, tsU46, tsU9 and ts1 in the DNA-binding domain are possibly not in the residues that are involved directly in DNA binding but have a profound effect on the conformation of the DNA-binding domain (Lim and Sauer, 1991
) [(the cI857ts mutation is known to replace Ala by Thr at 66th position in helix 4 of the DNA-binding domain (Sanger et al., 1982
)]. It is likely that the positions at which such changes of amino acids have occurred are very important for the maintenance of the functional conformation of the protein. It is interesting to know that the tsU51 mutation in the C-terminal domain has two changes, F141I and P153L (Table I
). It may be noted that
cItsU51 also shows the ind phenotype (Lieb, 1966
), but which of the above two mutations confers the ind property is not known. Beckett et al. (1993) isolated a non-cooperative mutation at position 147 (G147D) which is located in between the two U51 mutations. Although the crystal structure of the C-terminal domain of
repressor is not known, the structural importance of the residues may be guessed from the crystal structure of the homologous protein UmuD'. The residue F141 is semi-conserved (F or Y) among the four homologous proteins, UmuD', MucA, LexA and CI (Peat et al., 1996
). If the structure of UmuD' is used as a rough guide of CI C-terminal domain structure, then F141 appears to be removed from the clusters of non-cooperative mutant residues in CI. P153L, on the other hand, falls in a five amino acid insert that is unique to CI of
among the above four homologous proteins. This insert is adjacent to the residues the equivalent of which in UmuD' forms the only 310 helix in the structure. The short 310 helix in the UmuD' structure interacts with the region containing NASY reverse turn. The equivalent residues of non-cooperative mutations, Y210H, G147D and M212R, are located close to these structural elements. It may be that the above two U51 mutations destabilize this important region in a temperature-dependent manner and the conformation of the hinge region of this mutant protein is altered in such a way that the protein is not accessible to RecA, thereby imposing the ind property.
The N207T change by tsI-22 mutation appears to be very close to a predicted reverse turn around which some non-cooperative mutations have been isolated (Whipple et al., 1994). It has been argued that this region is involved in direct proteinprotein cooperative contact. Hence, the tsI-22 mutation may destabilize the region around position 207 at non-permissive temperatures. Recently, it has been shown that the non-cooperative mutant Y210C may exert its effect through indirect means (Deb et al., 1998
). This also suggests that N207 may not be directly involved in proteinprotein interaction, and its role may be in the formation of the putative reverse turn.
The importance of proteinprotein contact in negative regulation in prokaryote is now well appreciated. The exact role of such contact is, however, not clear. It certainly augments the strength of the proteinDNA interaction. However, a different role such as the transmission of the effect of conformational change may also be involved. Structural and functional studies of non-cooperative mutants may shed light on these aspects. The C-terminal domain of the ts2 repressor was found to have significant perturbation in the structure and possibly a weaker cooperativity even at permissive temperatures. This weakened cooperativity, however, is still sufficient to allow the establishment and maintenance of lysogeny to take place at permissive conditions. At higher temperatures, the structure appears to be severely disrupted, leading, perhaps, to a complete loss of cooperativity and a loss of the ability to lysogenize as well as to maintain lysogeny (Lieb, 1966). Even with the availability of the crystal structure, the nature of the monomermonomer interface of UmuD' remains controversial. The conclusions drawn about the nature of the monomermonomer interface from the NMR study of Ferentz et al. (1997) are not compatible with the interface seen in the crystal structure. The charge reversal by ts2 (K224E) mutation is dramatic. The residue K224 is located near the residue V222. Equivalent residues in UmuD' are part of a ß sheet and the side chains of these residues would be immediately adjacent. In the crystal structure of UmuD', F128, which is equivalent to V222 in
CI, is part of the monomermonomer interface. In the proposed NMR structure, however, the C-terminal tail region forms part of the monomermonomer interface and the residue corresponding to K224 is adjacent to this important interaction point. Therefore, it is possible that in either case K224 is close to the monomermonomer interface. Hence a charge reversal at this point exerts a strong destabilizing effect on proteinprotein interactions, causing impaired function particularly at higher temperatures. The observed effect of this mutation on the accessibility of several Trp and sulfhydryl residues clearly suggests that the destabilization is not confined to local dimensions.
Study of several non-cooperative mutant repressors (Burz and Ackers, 1994; Burz et al., 1994
) suggests that many of them have significantly enlarged hydrated volumes, indicating disruption of their native structure. Unfortunately, not many of them have been studied from a structural point of view. Study of the ts2 repressor clearly underlines the possibility that many of the mutants may have indirect functional effects and, hence, cannot be used to identify the regions of proteinprotein contact. Such ts mutants in many cases, however, clearly provide information about structural connectivities to important functional regions. Many non-cooperative and ts mutants are now known in the C-terminal domain of
repressor. Lack of a crystal or NMR structure makes it difficult to relate it to specific structural roles. The recent availability of a homologous UmuD' structure (Peat et al., 1996
), however, may allow one to speculate about the structural regions involved in the dimerdimer interaction. Equivalent residues of one group of non-cooperative mutation fall within a general area of the UmuD' structure. These mutations are G147D, E188K, K192N and M212R. The C
atom of the equivalent residue of G147 in UmuD' is within 6 Å of NH of the equivalent residue of N192 in UmuD'. Similarly, the equivalent residue of M212 of
CI, an isoleucine in UmuD', is within 8 Å of the equivalent residue of K192 in UmuD'. For another group of mutations in CI of
, the equivalent residues in UmuD' form the 310 helix and NASY reverse turn, which are close together in space. These are P153L (ts51), N207T (tsI-22) and Y210C or Y210H. The existence of ts mutants in this region strongly suggests that these mutations exert their influence through structural alteration at non-permissive temperatures and are not the interaction points as such. Our recent studies with Y210C also suggest that it is an indirect mutant. Equivalent residues of a third group of mutations, R196G, D197G, D197A, S198N, S198R, G199D, G199V and F202S, fall on an exposed loop in the UmuD' structure. Equivalent residues of a fourth group of mutations falls in the C-terminal tail side, which include S228N, T234K and, possibly, K224E (ts2). Both S228N and T234K have been shown to be defective in dimermonomer transition and are not likely to exert their effect on cooperativity, if any, by direct contact. K224E is unlikely to be a direct-contact mutation and probably exerts its effect through destabilization of the protein structure as revealed by spectroscopic studies (Figures 26
). The general destabilization seen in the ts2-repressor structure strongly argues in favor of this. Hence it appears likely that the regions around G147 and the 196199 loop may be the primary contact points for dimerdimer interactions.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bandyopadhyay,S., Banik,U., Mandal,N.C., Bhattacharyya,B. and Roy,S. (1995) Biochemistry, 34, 50905097.[ISI][Medline]
Banik,U., Saha,R., Mandal,N.C., Bhattacharyya,B. and Roy,S. (1992) Eur. J. Biochem., 206, 1521.[Abstract]
Banik,U., Saha,R., Mandal,N.C., Bhattacharyya,B. and Roy,S. (1993) J. Biol. Chem., 268, 39383943.
Beckett,D., Burz,D.S., Ackers,G.K. and Sauer,R.T. (1993) Biochemistry, 32, 90739079.[ISI][Medline]
Brack,C. and Pirrotta,V. (1975) J. Mol. Biol., 96, 139152.[ISI][Medline]
Bukau,B. and Horwich,A.L. (1998) Cell, 92, 351366.[ISI][Medline]
Burz,D.S. and Ackers,G. (1994) Biochemistry, 33, 34063416.
Burz,D.S., Beckett,D., Benson,N. and Ackers,G. (1994) Biochemistry, 33, 83998405.[ISI][Medline]
Das,T. and Mandal,N.C. (1986) Mol. Gen. Genet., 204, 540542.[ISI][Medline]
Deb,S., Bandyopadhyay,S. and Roy,S. (1998) Protein Engng, 11, 481487.[Abstract]
Ellman,G.L. (1959) Arch. Biochem. Biophys., 82, 7077.[ISI][Medline]
Ferentz,A.E., Opperman,T., Walker,G.C. and Wagner,G. (1997) Nature Struct. Biol., 4, 979983.[ISI][Medline]
Jordan,S.R. and Pabo,C.O. (1988) Science, 242, 893899.[ISI][Medline]
Kaiser,A.D. (1957) Virology, 3, 4261.[ISI][Medline]
Lieb,M. (1966) J. Mol. Biol., 16, 149163.[ISI][Medline]
Lieb,M. (1976) Mol. Gen. Genet., 146, 291298.[ISI][Medline]
Lim,W.A. and Sauer,R.T. (1991) J. Mol. Biol., 219, 359376.[ISI][Medline]
Mandal,N.C. and Lieb,M. (1976) Mol. Gen. Genet., 146, 299302.[ISI][Medline]
Mandal,N.C., Crochet,M. and Lieb,M. (1974) Virology, 57, 8592.[ISI][Medline]
Nag,D.K., Chattopadhyay,D.J. and Mandal,N.C. (1982) Virology, 118, 448450.[ISI][Medline]
Nag,D.K., Chattopadhyay,D.J. and Mandal,N.C. (1984) Mol. Gen. Genet., 194, 373376.[ISI][Medline]
Nelson,H.C.M. and Sauer,R.T. (1985) Cell, 42, 549558.[ISI][Medline]
Pabo,C.O. and Lewis,M. (1982) Nature, 298, 443447.[ISI][Medline]
Peat,T.S., Frank,E.G., McDonald,J.P., Levine,A.S., Woodgate,R. and Hendrockson,W.A. (1996) Nature, 230, 727730.
Ptashne,M. (1992) A Genetic Switch: Gene Control and Phage Lambda. Cell Press, Boston.
Ptashne,M., Jeffrey,A., Johnson,A.D., Maurer,R., Meyer,B.J., Pablo,C.O., Roberts,T.M. and Sauer,R.T. (1980) Cell, 19, 119.[ISI][Medline]
Riggs,A.D., Bourgeois,S., Newby,R.F. and Cohn,M. (1968) J. Mol. Biol., 34, 365368.[ISI][Medline]
Saha,R., Banik,U., Bandyopadhyay,S., Mandal,N.C., Bhattacharyya,B. and Roy,S. (1992) J. Biol. Chem., 267, 58625867.
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl Acad. Sci. USA, 74, 54635467.[Abstract]
Sanger,F., Coulson,A.R., Hong,G.F., Hill,D.F. and Peterson,G.B. (1982) J. Mol. Biol., 162, 729773.[ISI][Medline]
Sauer,R.T. and Anderegg,R. (1978) Biochemistry, 17, 10921100.[ISI][Medline]
Sussman,R. and Jacob,F. (1962) C. R. Acad. Sci., 254, 15171519.[ISI]
Whipple,F.W., Kuldell,N.H., Cheatham,L.A. and Hochschild,A. (1994) Genes Dev., 8, 12121223.[Abstract]
Received June 20, 1998; revised November 10, 1998; accepted December 7, 1998.