Purnima Bhargava

Histones in functional diversification

Recent research suggests that minor changes in the primary sequence of the conserved histones may become major determinants for the chromatin structure regulating gene expression and other DNA‐related processes. An analysis of the involvement of different core histone variants in different nuclear processes and the structure of different variant nucleosome cores shows that this may indeed be so. Histone variants may also be involved in demarcating functional regions of the chromatin. We discuss in this review why two of the four core histones show higher variation. A comparison of the status of variants in yeast with those from higher eukaryotes suggests that histone variants have evolved in synchrony with functional requirement of the cell.

Seminalplasmin and caltrin are the same protein

Seminalplasmin Caltrin Seminal plasma protein

Nucleosome positioning in relation to nucleosome spacing and DNA sequence-specific binding of a protein

Nucleosome positioning is an important mechanism for the regulation of eukaryotic gene expression. Folding of the chromatin fiber can influence nucleosome positioning, whereas similar electrostatic mechanisms govern the nucleosome repeat length and chromatin fiber folding in vitro. The position of the nucleosomes is directed either by the DNA sequence or by the boundaries created due to the binding of certain trans‐acting factors to their target sites in the DNA. Increasing ionic strength results in an increase in nucleosome spacing on the chromatin assembled by the S‐190 extract of Drosophila embryos. In this study, a mutant lac repressor protein R3 was used to find the mechanisms of nucleosome positioning on a plasmid with three R3‐binding sites. With increasing ionic strength in the presence of R3, the number of positioned nucleosomes in the chromatin decreased, whereas the internucleosomal spacings of the positioned nucleosomes in a single register did not change. The number of the positioned nucleosomes in the chromatin assembled in vitro over different plasmid DNAs with 1–3 lac operators changed with the relative position and number of the R3‐binding sites. We found that in the presence of R3, nucleosomes were positioned in the salt gradient method of the chromatin assembly, even in the absence of a nucleosome‐positioning sequence. Our results show that nucleosome‐positioning mechanisms are dominant, as the nucleosomes can be positioned even in the absence of regular spacing mechanisms. The protein‐generated boundaries are more effective when more than one binding site is present with a minimum distance of ∼ 165 bp, greater than the nucleosome core DNA length, between them.

The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein.

Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGal, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the TBP dimer levels bothin vitro andin vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity bothin vitro andin vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditionsin vitro. Usingin vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg2+, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg2+, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg2+, resulted in dimerization of TBP. Effect of Mg2+ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg2+. At both the levels, activity of the full-length TBP in the presence of Mg2+ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.

Spectroscopic studies on the mode of binding of ATP, UTP and α-amanitin with yeast RNA polymerase II

The binding affinity between the substrates ATP and UTP with the purified yeast RNA polymerase II have been studied here in the presence and absence of Mn2+. In the absence of template DNA, both ATP and UTP showed tight binding with the enzyme without preference for any specific nucleotide, unlike Escherichia coli RNA polymerase. Fluorescence titration of the tryptophan emission of the enzyme by nucleoside triphosphate substrates gave an estimated K d value around 65 μM in the absence of Mn2+ whereas in the presence of Mn2+, the K d was 20 μM. The effect of substrates on the longitudinal relaxation of the HDO proton in enzyme‐substrate complex also yielded a similar K d value.

Influence of substrate and template binding on the interaction of α-amanitin with yeast RNA polymerase II: Fluorescence spectroscopic analysis

Fluorescence titration of the tryptophan residues in yeast RNA polymerase II with α‐amanitin shows there are two types of binding between the inhibitor and the enzyme. When the stronger binding site was saturated with α‐amanitin, the enzyme‐inhibitor complex could not be further titrated efficiently with ATP, indicating that probably the inhibitor and the substrate binding domains on the enzyme are overlapping. Supercoiled plasmid DNA bearing alcohol dehydrogenase I promoter of yeast showed binding with purified yeast RNA polymerase II in the absence of substrates. However, when the enzyme‐inhibitor complex was titrated with this template, it was found that the complex behaves in a similar way as the enzyme alone towards the template DNA. It suggests that probably the inhibitor and the template binding sites on the enzyme are quite different.

Seminalplasmin inhibits transcription and translation of φ80 DNA in vitro

Seminalplasmin, an antimicrobial protein from bovine seminal plasma that has been earlier shown to inhibit transcription in whole cells and by purified RNA polymerase in vitro, but not translation in whole cells, is now shown to inhibit both transcription and translation independently of each other, in a coupled transcription‐translation system from E. coli using φ80dphoAlacZ DNA as the template.

DNA intervention in transcriptional activation.

Accurate initiation of eukaryotic mRNA synthesis takes place as a result of the interplay between general transcription factors and RNA polymerase II. Activation of transcription from the basal level involves a number of promoter‐specific trans‐acting factors which interact with cis elements in the promoter DNA. In this paper we have emphasized the importance of even those portions of the promoter stretch which do not have any identifiable binding sites for regulatory proteins. The length and structure of the DNA between cognate binding sites of trans‐acting factors may interfere with the level of transcriptional activation. Depending upon the length of the intervening DNA we describe three cases of transcriptional activation. In addition, based on this classification we propose a new third domain, the other two being DNA binding and transcriptional activation domains, which is involved in bending the intervening DNA so that activation from a distance can take place successfully.

Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

Many regulatory proteins and complexes influence transcription by RNA polymerase (pol) II. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding genes. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to identify potential interactors through mass spectrometry-based proteomics. A large interactome constituting known chromatin modifiers, factors and regulators of transcription by pol I and pol II revealed the possibility of a large number of signaling cues for pol III transcription against adverse conditions. We found one of the pol II-associated factors, Paf1 complex (PAF1C) interacts with the three baits. Its occupancy on the pol III-transcribed genes is low and not correlated with pol III occupancy. Paf1 deletion leads to higher occupancy of pol III, γ-H2A and DNA pol2 but no change in nucleosome positions. Genotoxins exposure causes pol III but not Paf1 loss from the genes. PAF1C promotes the pol III pausing and restricts its accumulation on the genes, which reduces the replication stress caused by the pol III barrier and transcription-replication conflict on these highly transcribed genes.