Bianca Sclavi

Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage

All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co‐ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at specific sites on the chromosome termed origins of replication, and proceeds bidirectionally to elongate and duplicate the chromosome. These two processes, initiation and elongation, therefore determine the total rate of DNA synthesis in the cell. In Escherichia coli, initiation depends on the DnaA protein while elongation depends on a multiprotein replication factory that incorporates deoxyribonucleotides (dNTPs) into the growing DNA chain. The enzyme ribonucleotide reductase (RNR) is universally responsible for synthesizing the necessary dNTPs. In this review we examine the role RNR plays in regulating the total rate of DNA synthesis in E. coli and, hence, in maintaining constant DNA/cell mass ratios during normal growth and under conditions of DNA stress.

Following the folding of RNA with time-resolved synchrotron X-ray footprinting.

The rapid mixing synchrotron X-ray footprinting technique described in this article allows nucleic acid folding and ligand binding reactions to be followed on a millisecond time resolution with single nucleotide resolution. In principle, the change in .OH protection of every nucleotide in a nucleic acid hundreds of nucleotides long can be monitored separately. In addition, a wide range of solution conditions are compatible with the radiolytic generation of .OH. These characteristics of synchrotron X-ray footprinting create opportunities for conducting thermodynamic and kinetic studies of nucleic acids that are both comprehensive and detailed. Kinetic footprinting studies of a number of systems have been initiated by the Center for Synchrotron Biosciences using this technique.

Microfluidic chemostat for measuring single cell dynamics in bacteria

We designed a microfluidic chemostat consisting of 600 sub-micron trapping/growth channels connected to two feeding channels. The microchemostat traps E. coli cells and forces them to grow in lines for over 50 generations. Excess cells, including the mother cells captured at the start of the process, are removed from both ends of the growth channels by the media flow. With the aid of time-lapse microscopy, we have monitored dynamic properties such as growth rate and GFP expression at the single-cell level for many generations while maintaining a population of bacteria of similar age. We also use the microchemostat to show how the population responds to dynamic changes in the environment. Since more than 100 individual bacterial cells are aligned and immobilized in a single field of view, the microchemostat is an ideal platform for high-throughput intracellular measurements. We demonstrate this capability by tracking with sub-diffraction resolution the movements of fluorescently tagged loci in more than one thousand cells on a single device. The device yields results comparable to conventional agar microscopy experiments with substantial increases in throughput and ease of analysis.

Time-resolved synchroton X-ray footprinting and its application to RNA folding

Publisher Summary This chapter discusses the application of synchrotron X-ray footprinting for studying RNA folding at beam line X-9A. Beam line X-9A is a bending magnet beam line producing white light over an energy range of 3–30 keV. The radiolysis reaction products are separated using polyacrylamide gel electrophoresis (PAGE). A storage phosphor screen and associated imager are used to acquire a digital image of the electrophoretogram for densitometric analysis. Band intensity is quantitated using the molecular dynamics image quant or equivalent image analysis software. The intensity within a protected region and a reference region is quantitated for each lane on the gel. The reference region is a set of bands that accounts for variability in sample loading in the individual lanes but that shows the same degree of protection throughout the folding process. The densitometric results are exported to a spreadsheet (Microsoft Excel) for further processing and analysis. Each protected region is divided by the corresponding reference region in the same lane. The resulting data points are plotted as a function of reaction time. The protection quantitated to obtain this curve represents bases 118–120 within the P4–P6 domain of the ribozyme and appears at a rate 0.2 sec −1 . This protection is believed to represent a tertiary contact with bases within the peripheral domain P9.1 that show a comparable rate of protection.

DnaA‐ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli

Ribonucleotide reductase (RNR) is the bottleneck enzyme in the synthesis of dNTPs required for DNA replication. In order to avoid the mutagenic effects of imbalances in dNTPs the amount and activity of RNR enzyme in the cell is tightly regulated. RNR expression from the nrdAB operon is thus coupled to coincide with the initiation of DNA replication. However, the mechanism for the co‐ordination of gene transcription and DNA replication remains to be elucidated. The timing and synchrony of DNA replication initiation in Escherichia coli is controlled in part by the binding of the DnaA protein to the origin of replication. DnaA is also a transcription factor of the nrdAB operon and could thus be the link between these two processes. Here we show that RNA polymerase can form a stable transcription initiation complex at the nrdAB promoter by direct interaction with the far upstream sites required for the timing of expression as a function of DNA replication. In addition, we show that the binding of DnaA on the promoter can either activate or repress transcription as a function of its concentration and its nucleotide‐bound state. However, transcription regulation by DnaA does not significantly affect the timing of expression of RNR from the nrdAB operon.

Examining the conformational dynamics of macromolecules with time-resolved synchrotron X-ray ‘footprinting’

This work is supported by grants from the National Center for Research Resources (RR-01633, MRC) and the National Institute for General Medical Sciences (GM-52348, MRC; GM-51506, M.B. & GM-46686, S.W.) of the National Institutes of Health. The operation of the NSLS is supported by the Office of Basic Energy Sciences, Department of Energy. We also thank Mike Sullivan for assistance in this project.

Gene clusters reflecting macrodomain structure respond to nucleoid perturbations.

Focusing on the DNA-bridging nucleoid proteins Fis and H-NS, and integrating several independent experimental and bioinformatic data sources, we investigate the links between chromosomal spatial organization and global transcriptional regulation. By means of a novel multi-scale spatial aggregation analysis, we uncover the existence of contiguous clusters of nucleoid-perturbation sensitive genes along the genome, whose expression is affected by a combination of topological DNA state and nucleoid-shaping protein occupancy. The clusters correlate well with the macrodomain structure of the genome. The most significant of them lay symmetrically at the edges of the Ter macrodomain and involve all of the flagellar and chemotaxis machinery, in addition to key regulators of biofilm formation, suggesting that the regulation of the physical state of the chromosome by the nucleoid proteins plays an important role in coordinating the transcriptional response leading to the switch between a motile and a biofilm lifestyle.

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing C-terminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.

DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37°C and results in the accumulation of an off-pathway intermediate

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild-type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus –10 region. Finally, the comparison of the results obtained at 37°C with those at 20°C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.

Gene Regulation by H-NS as a Function of Growth Conditions Depends on Chromosomal Position in Escherichia coli

Cellular adaptation to changing environmental conditions requires the coordinated regulation of expression of large sets of genes by global regulatory factors such as nucleoid associated proteins. Although in eukaryotic cells genomic position is known to play an important role in regulation of gene expression, it remains to be established whether in bacterial cells there is an influence of chromosomal position on the efficiency of these global regulators. Here we show for the first time that genome position can affect transcription activity of a promoter regulated by the histone-like nucleoid-structuring protein (H-NS), a global regulator of bacterial transcription and genome organization. We have used as a local reporter of H-NS activity the level of expression of a fluorescent reporter protein under control of an H-NS−regulated promoter (Phns) at different sites along the genome. Our results show that the activity of the Phns promoter depends on whether it is placed within the AT-rich regions of the genome that are known to be bound preferentially by H-NS. This modulation of gene expression moreover depends on the growth phase and the growth rate of the cells, reflecting the changes taking place in the relative abundance of different nucleoid proteins and the inherent heterogeneous organization of the nucleoid. Genomic position can thus play a significant role in the adaptation of the cells to environmental changes, providing a fitness advantage that can explain the selection of a gene’s position during evolution.