Beltcho G. Beltchev

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

Involvement of protein HMG1 in DNA replication

Antibodies against HMG1 inhibit the incorporation of [3H]thymidine in Ehrlich ascites cell nuclei. By the use of specific inhibitors it is shown that HMG1 is needed for the action of the replicative DNA polymerase and not for the reparative one. This is supported by the fact that the addition of exogenous HMG1 to the nuclei enhances the replication process.

Acetylated HMG1 protein interacts specifically with homologous DNA polymerase alpha in vitro.

The acetylated, deacetylated and nonacetylated forms of HMG1 proteins from Guerin ascites tumour cells and calf thymus were separated and their in vitro interactions with homologous and heterologous DNA polymerases were studied. It has been found that only the acetylated form of HMG1 proteins forms a specific complex with homologous DNA polymerase alpha and stimulates its activity in vitro. The acetylation therefore is necessary for their possible function in DNA replication. This finding represents an evidence for a relationship between the acetylation of HMG1 proteins and their biological role.

Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins

We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.

Preparation of a pG-fragment from tRNAPheyeast by chemical scission at the dihydrouracil, and inhibition of tRNAPheyeast charging by this fragment when combined with the -CCA half of this tRNA

Some insigl~t into the specificity of the recognition reaction of tRNA,synthetase was provided by studies with large fragments of tRNA [1 -4 ] . We are reporting here a chemical method to obtain a quarter of a tRNA Phe molecule (which we call pGfragment). This fragment, 15 nucleotides long, starting from the hU residue in position 16 of yeast tRNA Phe and ending at the pG terminus was isolated by a chain scission at the reduced hU. Charging of yeast tRNA Phe is competitively inhibited by the combination of this pG-fragment with the chemically obtained -CCA portion of yeast tRNA Phe, while each of these fragments, taken individually, has no effect.

Differences between some properties of acetylated and nonacetylated forms of HMG1 protein.

There are data suggesting that HMG1 protein may be involved in DNA replication. Recently we have found that only the acetylated form of the protein generates tetramers, stimulates the activity of DNA polymerase alpha (EC 2.7.7.7) (with activated DNA as a template) and forms a specific complex with it. This paper compares some properties of the acetylated and nonacetylated forms of HMG1 protein and shows that it is only the acetylated form which serves as a histone assembly factor, increases the melting temperature of poly d[(A-T)] and stimulates the activity of DNA polymerase alpha when histone H1-depleted chromatin is used as a template.

Differences between HMG1 proteins isolated from normal and tumour cells

The properties of the non-histone chromosomal high-mobility-group 1 (HMG1) proteins from rat liver and Guerin ascites tumour cells (GAT cells) were compared and showed the following differences: (1) five spots were missing in the peptide map of HMG1 from GAT cells in comparison with that of HMG1 from rat liver; (2) HMG1 from GAT cells was about 5-times more poly(ADP)-ribosylated; (3) HMG1 from GAT cells which was found acetylated in vivo and incorporated [14C]acetate in vitro, whereas no incorporation of the label was detected in HMG1 from rat liver; (4) HMG1 from GAT cells exhibited pronounced ability to form oligomers at physiological ionic strength, while HMG1 from rat liver was predominantly in monomeric form. This property of HMG1 from GAT cells was lost upon deacetylation.

Influence of high-mobility-group nonhistone chromosomal proteins 1 and 2 on the digestion of chromatin with micrococcal nuclease

Abstract The digestion of different chromatin preparations from calf thymus with micrococcal nuclease was studied in order to estimate the effect of high-mobility-group nonhistone chromosomal proteins 1 and 2 on the structure of chromatin. Chromatin selectively depleted of histone H1 was digested faster than chromatin depleted of H1 and most of the nonhistone proteins including HMG1 and 2. Reconstitution of the latter chromatin with purified HMG1 or 2 raised its sensitivity to the level of selectively H1-depleted chromatin. Addition of HMG1 or 2 to DNA also increased the rate of digestion. Thus HMG1 and 2 have a decondensing effect on chromatin via destabilization of the structure of its DNA. The results are discussed in view of the possible involvement of HMG1 and 2 in the structure of transcriptionally active or replicating chromatin.

Protein HMG1 is Different from a DNA Helix Unwinding Protein in Calf Thymus

A number of criteria were used—chromatography on columns with single-stranded and double-stranded DNA, electrophoresis, peptide analysis, immunological tests and thermal denaturation of DNA—to show that protein (high mobility group) HMG1 and an unwinding protein from calf thymus are two distinct, unrelated proteins. While both proteins are thought to be related to DNA replication this might involve different mechanisms of action.

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

Abstract In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.