Structure-function relationships in nucleosomal arrays containing linker histone H5.

To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence. The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber. Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation.

Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers.

To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers.

Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion.

Repressive effect on oligonucleosome transcription of the core histone tail domains.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from histone octamers and three different DNA species, two circular (pGEMEX-1 and pT207-18) and one linear (T7-207-18). pGEMEX is devoid of nucleosome positioning sequences, while in pT207-18 and T7-207-18 the region downstream of the promoter contains 18 tandem repeats of a 207 bp positioning sequence derived from the 5S RNA gene of the sea urchin Lytechinus variegatus. Elimination of the histone tails in the assembled oligonucleosomes by trypsin digestion is accompanied, in all three DNA species, by substantial increases in transcription efficiency, assayed at different KCl and MgCl2 concentrations, after allowing for the aggregation observed under certain conditions.

Transcriptional properties of oligonucleosomal templates containing acetylated (H3-H4)2 tetramers.

Direct chemical acetylation of an oligonucleosomal template for bacteriophage T7 RNA polymerase is accompanied by a substantial increase in its capability to support RNA synthesis. The template was assembled from a plasmid, containing a promoter and a terminator for T7 RNA polymerase, plus one (H3-H4)2 tetramer and two H2A.H2B dimers for each 200 base pairs of DNA.

Acetylation of histone H2A.H2B dimers facilitates transcription.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for T7 RNA polymerase, intact (H3.H4)2 tetramers, and either untreated or chemically acetylated H2A.H2B dimers.

Efficient transcription of a DNA template associated with histone (H3.H4)2 tetramers.

A histone-DNA transcription template has been assembled, by dialysis against decreasing salt concentrations, from pGEMEX-1 (4 kilobases), a plasmid containing a promoter for bacteriophage T7 RNA polymerase, and from isolated histone (H3.H4)2 tetramers. Electron microscopy after psoralen cross-linking shows that each histone tetramer protects approximately 80 base pairs of DNA from psoralen action and that, under the employed conditions, an average of 15 tetramer particles are assembled per DNA molecule.

Transcription of mononucleosomal particles acetylated in the presence of n-butyrate.

Although a correlation between chemical acetylation of the amino-terminal tails of core histones and stimulation of RNA synthesis has been reported for nucleosomal core particles (Piñeiro et al. (1991) Biochem. Biophys.

Effect of high mobility group proteins 14 and 17 on the structural and transcriptional properties of acetylated complete and H2A.H2B-deficient nucleosomal cores.

Acetylation of H2A.H2B-deficient nucleosomal cores, like that of the complete particles, causes a substantial increase in the efficiency of the particles as in vitro transcription templates. Binding of the high mobility group proteins 14 and 17 (HMG 14/17) to chemically acetylated nucleosomal particles, both complete nucleosomal cores and those lacking one of the two H2A.

Succinylation of histone amino groups facilitates transcription of nucleosomal cores.

Treatment of nucleosomal cores with succinic anhydride, which modifies preferentially the amino-terminal domains of core histones, takes place without dissociation of the particles. Low levels of modification, which cause small structural effects, are accompanied by substantial increases in the efficiency of the nucleosomal cores as in vitro transcription templates for RNA polymerase II. The transcriptional properties of the succinylated nucleosomal cores are similar to those of the acetylated particles (Piñeiro et al.

Yeast nucleosomal particles: structural and transcriptional properties.

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.

Interaction of RNA polymerase II with acetylated nucleosomal core particles.

Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis.

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures.

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH. In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of diassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles.

Structural and transcriptional properties of different nucleosomal particles containing high mobility group proteins 14 and 17 (HMG 14/17).

Binding of high mobility group (HMG) proteins 14 and 17 (HMG 14/17) to complete nucleosomal cores and to cores lacking one H2A.H2B dimer, the amino-terminal tails of histones, or both one H2A.H2B dimer and the amino-terminal ends of histones is accompanied by an overall stabilization of the particles as determined by thermal denaturation, circular dichroism and DNase I digestion.

Reconstitution of rat liver 60S ribosomal subunits following disassembly by dimethylmaleic anhydride.

Modification of 60S ribosomal subunits from rat liver with dimethylmaleic anhydride (60 mumols/ml) is accompanied by release of 35% of the protein. The acidic ribosomal proteins, as well as 9 basic proteins, are selectively liberated from the ribosomal subunits. Reconstitution of the protein-deficient particles with the corresponding split proteins is accompanied by substantial recovery of the original polyphenylalanine synthetic activity.

Interaction of RNA polymerase II with structurally altered nucleosomal particles. Transcription is facilitated by loss of one H2A.H2B dimer.

The loss of one H2A.H2B dimer from the nucleosomal core increases its affinity for RNA polymerase II and its efficiency as a transcription template, allowing transcription of the entire DNA present in the particle. In contrast, the nucleosomal core lacking the amino-terminal ends of histones, which has an affinity for polymerase equal to that of the H2A.

Disassembly and reconstitution of yeast 60S ribosomal subunits.

Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mumol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins.

Dimethylmaleic anhydride, a specific reagent for protein amino groups.

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.

Preparation and structural characterization of nucleosomal core particles lacking one H2A.H2B dimer.

Nucleosomal core particles lacking one H2A.H2B dimer, (H2A.H2B)1 (H3.

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues.

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups.

Interaction with RNA polymerase of nucleosomal cores lacking one H2A.H2B dimer.

Nucleosomal particles lacking one H2A.H2B dimer interact with RNA polymerase from Escherichia coli more strongly than the complete nucleosomal core particles. Moreover, the in vitro transcription of the H2A.

Involvement of lysine and arginine residues in the binding of yeast ribosomal protein YL3 to 5S RNA.

The contribution of lysine and arginine residues to the formation of yeast ribonucleoprotein complex 5S RNA. protein YL3 has been investigated by determining the effects on complex formation of modification with chemical reagents specific for either lysine or arginine. Treatment of protein YL3 with acetic anhydride, maleic anhydride or phenylglyoxal is accompanied by loss of its capacity to bind to 5S RNA.

Pitfalls in the use of carboxylic acid anhydrides for structural studies of nucleoprotein particles.

During modification of protein amino groups with carboxylic acid anhydrides, these reagents cause a fall in pH, which can be prevented by addition of base. Although unmodified nucleosomal particles are not affected by the local transient changes in pH induced by the base (NaOH) added to prevent a fall in pH during modification, the nucleosomal particles modified by acetic anhydride are dissociated, with release of single-stranded DNA.

Peptidyl transferase centres of rat and yeast ribosomes. Different response to modification of protein amino groups.

Modification of rat liver ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, causes a large stimulation of peptidyl transferase activity assayed by the "fragment" reaction, as well as the inactivation of poly(U)-directed polyphenylalanine synthesis. In contrast to rat ribosomes, the peptidyl transferase of yeast ribosomes is little affected by modification. Although other interpretations are not excluded, these results might be due to differences between the peptidyl transferase centres of mammalian and yeast ribosomes.

Effects of different amino-group reagents on ribosomal integrity: structural role of lysine residues.

Treatment of 60S subunits from yeast ribosomes with dicarboxylic acid anhydrides (maleic, dimethylmaleic and tetrahydrophtalic), which introduces negatively-charged residues, is accompanied by substantial dissociation of protein components (35-55%). In contrast, acetic anhydride or cyanate, which introduce uncharged groups, cause practically no protein release, even after extensive modification. Therefore, in addition to blocking lysine-RNA interactions, a large change in the electric charge of the proteins appears to be necessary to obtain dissociation.