STRUCTURAL-ANALYSIS OF TRYPANOSOMA-BRUCEI-BRUCEI CHROMATIN BY LIMITEDPROTEOLYSIS - AN ELECTRICAL-BIREFRINGENCE STUDY

The sensitive electric-birefringence method was used to reveal structu ral differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orienta tion of the nucleosomal chains and the presence of extended DNA were a nalysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility ) were studied in relation to the orientational relaxation times. In c ontrast to the higher eukaryotes, the birefringence of T. brucei bruce i is negative and of low amplitude, corresponding to that of H1-deplet ed rat liver nucleosomes. Furthermore, the relaxation times are very s mall, about 10 mus. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA-protein interaction than for the high er eukaryotes. However, this condensation does not induce the formatio n of regular higher-order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1. The accessibility and structural role of histone-like proteins in T. bruce i brucei chromatin were also investigated using limited proteolysis wi th enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtili sin shows that proteins a and d, which are classified as H3 and H4 his tones, respectively, are the first to be attacked. The changes in chro matin conformation indicate that chromatin undergoes a structural tran sition, leading to decondensation, as indicated by increases in negati ve birefringence and relaxation time, and to a change in its orientati on mechanism, indicated by the appearance of a permanent moment. This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its involvement in nucleosome stabilization rather than higher-ord er condensation. Therefore, in T. brucei brucei chromatin, the charact eristic properties of proteins a and d (their composition and interact ion with DNA), as well as their external location on the nucleosome su rface, suggest that if these proteins play a role similar to that play ed by H3 and H4 in higher eukaryotes, probably through their N-termina l regions and interaction either with DNA or protein domains, the mech anisms involved in chromatin compaction are quite different. Since the re is no binding with H1, it may be hypothesized that a (non-histone) protein, or core protein domains, may play a part in chromatin compact ion and gene expression regulation comparable to that of H1 in higher eukaryotes.