In the first half of this thesis, we describe our study of the elongation dynamics of E. coli RNA polymerase using optical tweezers. Optical tweezers constitute an important tool in modern biophysical research, as they allow the manipulation and tracking of individual molecules, such as enzymes that carry out diverse biological functions by converting chemical energy into mechanical work. Improvements to the spatio-temporal resolution and accuracy of optical tweezers therefore directly impact our ability to probe the tiniest and fastest motions of such enzymes.

RNA polymerase is a central enzyme present in all organisms, that transcribes the genetic information encoded in DNA into RNA, one nucleotide at a time. This process constitutes the first step of gene expression, and is highly regulated at all its stages: initiation, elongation, and termination. In particular, elongation—i.e., the processive polymerization of the nascent RNA chain—does not occur in a continuous fashion, but consists of periods of active translocation interspersed by long-lived, sequence-dependent pauses, that have been implicated in various biological roles.

While optical tweezers have long been able to observe such long-lived pausing events, many questions remained open, due to the limited spatio-temporal resolution of the technique. Here, we demonstrate algorithmic and instrumental developments that improve our ability to probe the transcription cycle at the finest level. Improvements in spatial resolution allowed us to robustly observe individual translocation events over long distances, and thus record the distribution of the dwell times spent at each position by the enzyme. Improvements in temporal resolution and spatial accuracy allowed us to understand the dynamics of the enzymes immediately as it reaches a "pause site". Specifically, we were able to show that transcription through a pause site is always accompanied by a decrease of the forward transcription rate. We established that entry into "backtracked" pauses occurs in a stepwise fashion, with a relatively slow entry into deeply backtracked states. We also probed the effect of nascent RNA structures on RNAP dynamics, and found that, depending on the sequence context, such structures could either enhance or attenuate pre-existing pauses.

In the second half of this thesis, we review another fundamental single-molecule technique: super-resolution microscopy. Unlike optical tweezers, optical microscopy allows us to observe cellular processes in vivo or in situ; and the recent development of super-resolution microscopy has greatly enhanced the field of application of the technique. However, super-resolution microscopy also yields data that is much more difficult to interpret than classical ("diffraction-limited") microscopy. We discuss recent developments in our ability, not only to localize molecules with high accuracy, but also to quantify them. Finally, we present a fluorescent protein engineering work, regarding the development of a split-photoactivatable fluorescent protein system, towards the goal of studying protein-protein interaction at high resolution.