Chromosome metamorphoses: on the atypical roles of condensin II

Every human being originates from a single fertilized egg cell, continuously duplicating to form a body consisting of trillions of cells in a process known as the cell cycle. This cycle involves two major stages: interphase, where the cell prepares for division, and mitosis, where it divides into two daughter cells. The accurate duplication and...

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using...

High-throughput single-molecule experiments reveal heterogeneity, state switching, and three interconnected pause states in transcription

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high...

Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity...

SMC complexes can traverse physical roadblocks bigger than their ring size

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no...

Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers

Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing...

Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the...

Genome analysis and engineering of industrial lager brewing yeasts

Lager beer, also referred to as Pilsner, is the most popular alcoholic beverage in the world, with an annual consumption of almost 200 billion litres per year. To make lager beer, brewer’s wort is fermented with the yeast <i style="mso-bidi-font-style:normal"...

Slow unloading leads to DNA-bound ?2-sliding clamp accumulation in live Escherichia coli cells

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli ?2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both ?2-clamp...