Jean Pouyet

The carboxy-terminal domain of the LexA repressor oligomerises essentially as the entire protein.

The ability of the isolated carboxy‐terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85–202, is readily purified after self‐cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy‐terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non‐linear least squares fitting of the experimental concentration distribution. A dimer association constant of K 2 = 3 × 104 M−1 and a tetramer association constant of K 4 = 2 × 104 M−1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812–2818] gave values of K 2 = 2.1 × 104 M−1 and K 4 = 7.7 × 104 M−1. Within experimental error the dimer formation constant of the carboxy‐terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K 4 is higher than that in K 2. The overall conclusion is that the two structural domains of LexA have also well‐defined functional roles: the amino‐terminal domain interacts with DNA and the carboxy‐terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.

Spectroscopic studies of (m5dC-dG)3: thermal stability of B- and Z-forms.

The hexanucleoside pentaphosphate d(m5CpGpm5CpGpm5CpG) has been studied in solution by ultra-violet absorption, circular dichroism and 31P nuclear magnetic resonance under various experimental conditions. In 0.2 M NaClO4 at low temperature, an hexamer duplex is formed which has a B or B-like conformation. As the salt concentration is increased, a transition from a B-form to the Z-form occurs and is complete in 3 M NaClO4. In 3 M NaClO4, the behavior of the Z double helix is complex as a function of temperature. The variation of the circular dichroism at 295 nm is biphasic. A first transition occurs over a large range of temperature and corresponds to a conformational change due to a non-cooperative intramolecular process. Ultra-violet absorption and 31P nuclear magnetic resonance show that the new conformation arising from a distortion of the backbone is not similar to that observed in low salt conditions (B-form). At high hexanucleotide concentration, aggregates are formed. The second transition is cooperative and corresponds to the melting of a double stranded helix into single strands.

Core particle stability critically depends upon a small number of terminal nucleotides.

An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.

The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy

Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure. This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt. The use of urea or other denaturing agents and conditions is avoided. A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented. The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure. This method provides a more reliable analysis, especially of the contribution from beta-sheet. The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse. The latter group shows a larger contribution from beta-sheet. The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea. In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins. In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error. The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.

Ultracentrifugation studies of yeast valyl-tRNA synthetase and of its interaction with tRNAVal

Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 +/- 1500 and a sedimentation coefficient (SO 20, w) of 6.3 +/- 0.3 were found for the native enzyme. When the enzyme (3--60 muM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amounts of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA/enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant from tRNA/enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0--7.5) and in various buffers.

Kinetics of Nucleosomal Histone H1 Hyper(ADP-Ribosylation) and Polynucleosomes Relaxation

Poly(ADP-ribose) polymerase, a chromatin-bound enzyme, catalyzes postsynthetic modifications of various nuclear proteins through the covalent attachment of ADP-ribose units at the expense of the cellular NAD pool [1–3]. Poly(ADP-ribosylation) appears to be involved in DNA excision repair, cellular proliferation, and differentiation [1–3] and has been shown to induce architectural changes in chromatin [4–6].

Poly(ADP-Ribose) Glycohydrolase Activity Causes Recondensation of Relaxed Poly(ADP-Ribosyl)ated Polynucleosomes

Polyadenosine diphosphate ribose poly(ADP-ribose) polymerase catalyzes the incorporation of the ADP-ribose moiety of NAD into a homopolymer of repeating ADP-ribose units covalently bound to histones and other nuclear protein acceptors [1, 2]. This DNA-dependent enzyme, highly stimulated by nicks and DNA fragmentation [3, 4], is thought to be involved in several basic functions of the chromatin, especially in DNA repair [5–7].

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 μl), and the short dead time (∼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 K B k 2, 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.