Giuseppe Lia

Single-Molecule Approach to Molecular Biology in Living Bacterial Cells

Recent developments on fluorescent proteins and microscopy techniques have allowed the probing of single molecules in a living bacterial cell with high specificity, millisecond time resolution, and nanometer spatial precision. Recording movies and analyzing dynamics of individual macromolecules have brought new insights into the mechanisms of many processes in molecular biology, such as DNA-protein interactions, gene regulation, transcription, translation, and replication, among others. Here we review the key methods of single-molecule detection and highlight numerous examples to illustrate how these experiments are contributing to the quantitative understanding of the fundamental processes in a living cell.

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells

Dynamic Replication In all organisms, DNA replication involves a multiprotein complex called the replisome. Active Escherichia coli replisomes contain three copies of DNA polymerase III (Pol III). Lia et al. (p. 328, published online 22 December) used single-molecule spectroscopy to probe the dynamics of single proteins during E. coli replication in vivo. The results confirmed the presence of three Pol III molecules in the active replisome, with regular exchange of one of these Pol IIIs with one from the pool. Coordination with single-stranded DNA content suggests the Pol III that performs lagging-strand synthesis is exchanged so that a new Pol III is used for the synthesis of each Okazaki fragment. Single-molecule microscopy suggests that a new core polymerase is used to synthesize each Okazaki fragment. DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.

Statistical determination of the step size of molecular motors

Molecular motors are enzymatic proteins that couple the consumption of chemical energy to mechanical displacement. In order to elucidate the translocation mechanisms of these enzymes, it is of fundamental importance to measure the physical step size. The step size can, in certain instances, be directly measured with single-molecule techniques; however, in the majority of cases individual steps are masked by noise. The step size can nevertheless be obtained from noisy single-molecule records through statistical methods. This analysis is analogous to determining the charge of the electron from current shot noise. We review methods for obtaining the step size based on analysing, in both the time and frequency domains, the variance in position from noisy single-molecule records of motor displacement. Additionally, we demonstrate how similar methods may be applied to measure the step size in bulk kinetic experiments.

The antiparallel loops in gal DNA.

Interactions between proteins bound to distant sites along a DNA molecule require bending and twisting deformations in the intervening DNA. In certain systems, the sterically allowed protein–DNA and protein–protein interactions are hypothesized to produce loops with distinct geometries that may also be thermodynamically and biologically distinct. For example, theoretical models of Gal repressor/HU-mediated DNA-looping suggest that the antiparallel DNA loops, A1 and A2, are thermodynamically quite different. They are also biologically different, since in experiments using DNA molecules engineered to form only one of the two loops, the A2 loop failed to repress in vitro transcription. Surprisingly, single molecule measurements show that both loop trajectories form and that they appear to be quite similar energetically and kinetically.

RecA-Promoted, RecFOR-Independent Progressive Disassembly of Replisomes Stalled by Helicase Inactivation

In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.

ATP-dependent looping of DNA by ISWI

Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF-II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP-dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI.

Retraction. Polymerase exchange during Okazaki fragment synthesis observed in living cells.

Response MAYR QUESTIONS THE plausibility of our hypothesis that structural color signaling was the initial selective advantage in the evolution of pennaceous feathers. Our hypothesis is grounded in the accepted phylogenetic framework for theropods, which shows that pennaceous feathers evolved before flight (1–3). We thus disagree with Mayr’s suggestion that non-avian, nonflying theropods such as oviraptorosaurs inherited pennaceous feathers from their flying ancestors. Mayr argues that the loss of feather vane integrity in flightless birds indicates that pennaceous feathers evolved for the purpose of flight. However, according to Prum (4), the reduced integrity of the vane in most flightless birds is a variation of the original ancestral pennaceous feather. Mayr then argues that signaling feathers are localized, whereas in modern birds pennaceous feathers cover the entire body. Yet human visual capability strongly differs from that of birds, and biologically relevant optical cues are not restricted to the plumage that attracts our eye (5). Furthermore, the first known non-avian dinosaurs with planar feathers exhibited them in limited density only on prominent portions of the body (2), consistent with a signaling function. We agree with Mayr that the “wingassisted incline running” hypothesis (6) would provide an aerodynamic context for the evolution of pennaceous feathers, but the feathers evolved long before the potential for such behavior. Solitary planar feathers on the anterior extremities could have been gradually added, with respect to more sophisticated signaling, and at the same time incrementally forming lift-generating surfaces: the prerequisite for the transitory stages that resulted in active flight. Finally, Mayr points out that signaling feathers are “almost always” restricted to one sex, making an initial signaling function difficult to explain. However, intrasexual communication is not fully understood, especially in females (7). We should also context to streamline the body and form lift-generating surfaces. Based on the occurrence of essentially modern-type pennaceous feathers in a number of flightless non-avian theropod (“beast-footed”) dinosaurs, Foth et al. recently proposed that these feathers originated in a context other than flight (1). In their Perspective “Beyond the rainbow” (24 October, p. 416), M.-C. Koschowitz et al. argue that vaned feathers evolved for signaling with structural coloration. This hypothesis is, however, highly implausible

The manipulation of single biomolecules

Abstract By monitoring the response of individual protein and DNA molecules to pulling and twisting, biophysicists can learn much about their structure and their interactions. In this review we aim to give a flavour of the kinds of investigations that single-molecule techniques have made possible.