Search PubMed⌕ Search

Biomedical subjects

M Schnarr

Publications and source records attributed to M Schnarr.

54 records · Page 3Linked to original sources
PubMed1988

Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA.

Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA. These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation. This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface. The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers. Each half-operator contains seven interfering phosphates. These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target. On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis. We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein. A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain. We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.

Alkylation↗
PubMed1988

Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting.

A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes. The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A. After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation. Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase. The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt. A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator. The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment.

Bacterial Proteins↗
PubMed1987

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques.

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.

Bacterial Proteins↗
PubMed1987

Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain.

A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken. Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity. The presence of repressor increases the lag time, tau, without affecting the final maximum activity. The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state. A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer. Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor. The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species. These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction. Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene. The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.

Bacterial Proteins↗
PubMed1986

In vitro study of the interaction of the LexA repressor and the UvrC protein with a uvrC regulatory region.

The in vitro interaction of the LexA repressor with a regulatory region of the uvrC gene has been studied by polyacrylamide gel electrophoresis. Although the uvrC promoter region shows some homology with the canonic LexA binding site, no specific binding of the repressor to this DNA sequence could be observed, but only a cooperative nonspecific binding. By the same technique we show that the UvrC protein does not bind specifically to this regulatory DNA sequence either, although the protein is able to bind nonspecifically and cooperatively to the double-stranded DNA fragment.

Bacterial Proteins↗
PubMed1986

Fluorescence study of the RecA-dependent proteolysis of LexA, the repressor of the SOS system in Escherichia coli.

The fluorescence of the LexA protein, the common repressor of the SOS system in Escherichia coli decreases by about 30% upon incubation with the RecA protein, and its cofactors ATP [or its non-hydrolysable analogue adenosine-5'-O-(3-thiotriphosphate), ATP gamma S] Mg2+ and single-stranded DNA. In the absence of any one of these elements required for the RecA-dependent proteolysis of LexA, this fluorescence change was not observed. The final fluorescence change depends only upon the concentration of LexA regardless of that of RecA. The time course of the fluorescence decrease corresponds well with the kinetics of the decrease of intact LexA protein and the increase of its 2 proteolytic fragments as determined by SDS-polyacrylamide gel electrophoresis. These results allow us to use the fluorescence change as a signal for a detailed kinetic analysis. The velocity of the proteolysis (d[LexA]/dt) is proportional to the concentration of LexA and RecA indicating that the formation of the LexA-RecA complex is the limiting step.

Adenosine Triphosphate↗
PubMed1986

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Both the amino-terminal and the carboxy-terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA-catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non-specific binding to DNA, the purified amino-terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3-fold if the DNA lattice is fully saturated with protein. Further, the amino-terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino-terminal domain harbours a substantial amount of residues in alpha-helical conformation, a prerequisite for DNA recognition via a helix--turn--helix structural motif as proposed for many other regulatory proteins.

Bacterial Proteins↗
PubMed1985

Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli.

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.

Bacterial Proteins↗
PubMed1984

Effect of superhelicity on the transcription from the tet promoter of pBR322. Abortive initiation and unwinding experiments.

Supercoiling of DNA is now known to have considerable effects on transcription in bacteria. By abortive initiation reaction (6) we have determined the binding constant KB and the forward rate of isomerization k2 as a function of temperature, pH and buffer for the tet promoter in a supercoiled plasmid. If the activation energy of isomerization is very similar to that obtained previously under the same conditions on a linearized plasmid (6) (respectively 21 +/- 5 kcal/mole and 13 +/- 5 kcal/mole) the supercoiling introduces very important and not well understood changes in the thermodynamic parameters of the association polymerase - promoter. Using the technique of superhelical DNA relaxation by eukaryotic topoisomerase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430 degrees). This unwinding differs only slightly from the mean value (470 degrees) obtained for all the promoters of pBR322.

DNA, Superhelical↗
PubMed1984

Cooperative and salt-resistant binding of lexA protein to non-operator DNA.

The interaction of the lexA repressor of E. coli with poly[d(A-T)] has been studied by circular dichroism. The binding induces an about 2-fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid. The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly[d(A-T)] and natural DNA. At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.

Bacterial Proteins↗
PubMed1983

Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies.

The nonspecific interaction of the short headpiece, the NH2-terminal domain of the lac repressor, with natural DNA and alternating polydeoxynucleotides has been studied by means of fluorescence and circular dichroism. The important quenching of the intrinsic tyrosine fluorescence of the headpiece upon complexation has been used for the determination of the binding isotherms under various environmental conditions. By comparison with theoretical binding curves, we have determined a physical site size of three base pairs. The "perturbated" site size as determined from circular dichroism measurements (about four base pairs) is slightly greater. As in the case of the entire lac repressor, the interaction is strongly ionic strength dependent. The plots log Kobsd as a function of log [NaCl] are linear for salt concentrations greater than 50 mM for the interaction with DNA and greater than 25 mM for the interaction with poly[d(G-C)]. From the slopes of the linear parts of these plots, we determine a number of three electrostatic interactions, assuming no anion release from the protein upon complexation. This value is independent of pH. On the contrary, the association constant Kobsd depends on pH. Complexation requires the protonation of one titrating group of the headpiece with a pK value of 6.7 +/- 0.3, probably the residue histidine-29. The finding is in favor of the idea that the lac repressor interacts with DNA via two headpieces as earlier work has shown that the interaction of the lac repressor with DNA requires the protonation of two groups of the protein.

Circular Dichroism↗
PubMed1983

Binding of lac repressor headpiece to poly[d(A-T)]. A thermal denaturation study.

The binding of the lac repressor headpiece to poly[d(A-T)] has been investigated using thermal denaturation experiments. The binding of the headpiece leads to a stabilization of the double-stranded structure. Competition experiments using circular dichroism measurements confirm that the headpiece binds stronger on the double-stranded poly[d(A-T)] than on a single-stranded nucleic acid like poly(A). Theoretical analyses of the melting curves allow the determination of the binding constant and of the site size on the poly[d(A-T)]. This latter value is found to be 4 base pairs, in good agreement with that determined by other experimental approaches, and is much smaller than the values previously found for the lac repressor.

Circular Dichroism↗
PubMed1982

Secondary structure of the lac repressor headpiece. Possibilities and limitations of a joint infrared and circular dichroism study.

The secondary structure of the short tryptic headpiece of the lac repressor has been investigated by the analysis of its infrared and circular dichroic spectra. For the latter we used the method of Provencher and Glöckner [Biochemistry (1981) 20, 33-37], which seems to be at present the most successful for the determination of the beta content of proteins. Nevertheless our results indicate that in the case of the lac repressor headpiece this method overestimates the amount of beta structure. We find that the headpiece contains an important helical content of about 50%, depending slightly on the ionic strength. A decomposition of the infrared spectrum in a sum of Gaussian curves reveals clearly the absence of a vibrational band around 1630 cm-1, excluding thus the presence of a multi-stranded beta-pleated sheet. The only beta structure compatible with the infrared results seems to be a two-stranded antiparallel beta sheet, as judged from our results on the beta-sheet model-compound gramicidin S. The unusually strong intensity of the amide I' band is in favour of the existence of such a structure. The quantitative analysis of both infrared and circular dichroism spectra indicates the presence of a certain (but different) amount of beta structure. Comparing these results with several secondary structure predictions, part of the helical residues should be located between Leu-45 and (at least) Arg-35, and an eventual two-stranded beta sheet should be situated in the N-terminal part of the headpiece.

Circular Dichroism↗
PubMed1982

Stability of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis.

The stability of the conformation of the lac repressor headpiece against thermal denaturation and tryptic hydrolysis has been studied by circular dichroism measurements. In both cases the stability depends strongly on the concentration of NaCl. This effect is larger than generally observed for proteins. The midpoint of the thermal denaturation curve (Tm) is shifted from about 37 degrees C in the absence of NaCl to about 68 degrees C in 1 M NaCl. After a first non-linear increase of the Tm with the NaCl concentration (up to about 0.2 M NaCl) the Tm varies linearly with the salt concentration. Assuming a two-state mechanism for the thermal denaturation, enthalpies of 30-36 kcal/mol have been determined. The decrease of the circular dichroism signal due to the tryptic cleavage follows pseudo first-order kinetics for all salt concentrations studied. The half-life time of hydrolysis increased by about 40-times from 2 mM to the highest NaCl concentration we have used (655 mM). Assuming that only the unfolded state of the headpiece is a good substrate for trypsin, the observed stabilization against proteolytic degradation may be explained by a shift of the unfolding equilibrium of the headpiece due to the salt, and a subsequent decrease of the concentration of the unfolded state. The unusual stabilization of the headpiece is discussed with respect to its positive charge and to its function to bind to DNA.

Circular Dichroism↗
PubMed1982

Interaction between the lac operator and the lac repressor headpiece: fluorescence and circular dichroism studies.

The interaction between the lac repressor headpiece and a small operator DNA fragment has been examined by fluorescence and circular dichroism (c.d.) measurements. Binding of the headpiece to the DNA fragment induces a strong quenching of the fluorescence of its tyrosine residues. Quantitative analysis of the fluorescence data demonstrates that, in a first step, two headpieces bind very strongly to the DNA fragment then weaker binding occurs. C.d. demonstrates that the binding induces conformational changes of the DNA. The c.d. change produced upon binding of the first two headpieces differs from that induced upon binding of two further headpieces . Binding of the second pair of headpieces is similar to non-specific binding to non-operator DNA. The conformation of the operator DNA in the presence of two headpieces differs drastically from that in presence of lac repressor. Addition of the core to the lac operator does not induce any conformational change of the nucleic acids. These results are discussed with respect to the relative roles of core and headpieces in the lac repressor-lac operator interaction.

Circular Dichroism↗
PubMed1981

Unfolding of lac repressor and its proteolytic fragment by urea: headpieces stabilize the core within lac repressor.

Circular dichroism measurements were used to compared the urea-induced unfolding transition of the lac repressor with those of its separated tryptic fragments, the tetrameric core, and the N-terminal headpiece. The presence of the headpieces covalently linked to the core in the intact repressor leads to a stabilization against urea denaturation as compared to that for the isolated core. This results in a shift of the midpoint of the transition by about 0.5 M urea. When the inducer isopropyl beta-D-thiogalactoside is bound, the core is stabilized more than the entire repressor. The isolated headpiece is considerably more stable against urea denaturation than the tryptic core or the lac repressor. The reversible denaturation process of the headpiece was quantitatively analyzed, and the free energy of unfolding in the absence of urea was found to be 2.4 or 2.9 kcal/mol, depending on the method of calculation used. Comparison between the circular dichroism spectra of the lac repressor, the tryptic core of the lac repressor, and the headpiece supply further evidence that there are no major conformational differences between the structural domains (core and headpieces) before and after proteolytic cleavage of the lac repressor. These results are discussed with respect to the contacts between the different domains of the protein. It is concluded that relatively weak interdomain contacts are probably responsible for the stabilization of the core by the covalently linked headpieces and that these contacts might be weakened upon binding of the inducer.

Circular Dichroism↗
PubMedDate not supplied

A c-Jun activation domain peptide and its corresponding phosphopeptide have potential to adopt alpha-helical conformation.

The c-Jun transcription factor contains a transactivation domain that belongs to the "acidic" type of transcription activator. To determine the secondary structure of the Jun activation domain, we synthesized a peptide corresponding to amino acids 61 to 98 of c-Jun. Jun N-terminal kinases are able to phosphorylate Ser-63 and Ser-73 in vivo, which dramatically increases the transactivation potential of Jun. As this phosphorylation event may influence the secondary structure, we synthesized a second peptide containing two phosphoserine groups instead of serine in positions 63 and 73. Secondary structure predictions did not show potential for the peptides to adopt any stable, dominating conformation. Both peptides were purified and analyzed by circular dichroism spectroscopy. The peptides appeared to be flexible and essentially unstructured in aqueous solution. At acidic pH, we observed a decrease in the negative ellipticity at 202 nm, suggesting that some ordered structure might be present under these conditions. alpha-Helical conformation, as a dominating secondary structure, was induced in the presence of trifluoroethanol, and there was no significant difference between the unphosphorylated and phosphorylated peptides.

Circular Dichroism↗