Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.

We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

Topoisomerases inhibition and DNA binding mode of daunomycin-oligoarginine conjugate.

Cancer is a major health issue adsorbing the attention of a biomedical research. To fight this disease, new drugs are developed, specifically tailored to target biological pathways or peculiar components of the tumour cells. Particularly interesting is the use of intercalating agents as drugs capable to bind DNA and inhibit enzymes involved in DNA metabolism. Anthracyclines are the most commonly used anticancer drugs. In particular, daunomycin is used to cancer treatment by exploiting its ability to intercalate DNA and inhibit the activity of DNA topoisomerases implicated in the replication processes. Unfortunately, clinical application of anthracyclines is limited by their side effects. The conjugation with specific carriers could affect the selectivity and reduce side effect by improving stability and/or cellular uptake properties. We here report the biochemical characterisation of a daunomycin oligopeptide conjugate containing six residues of arginine, by the analysis of its fluorescence properties, DNA interaction and topoisomerases inhibitory effects.

Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

Structure and Properties of DNA Molecules Over The Full Range of Biologically Relevant Supercoiling States.

Topology affects physical and biological properties of DNA and impacts fundamental cellular processes, such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may change according to physiological and environmental conditions. Elucidation of structural properties of DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of positive and negative plasmid molecules.

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.