Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical–accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Summary: 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.

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.

We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37°C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the α-subunit C-terminal domain and binding of the σ subunit to the -35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the -10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the β/β′ jaws, leading to a transcriptionally active complex.