A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Preparation of Bioderived and Biodegradable Surfactants Based on an Intrinsically Disordered Protein Sequence.

Surfactants, block copolymers, and other types of micellar systems are used in a wide variety of biomedical and industrial processes. However, most commonly used surfactants are synthetically derived and pose environmental and toxicological concerns throughout their product life cycle. Because of this, bioderived and biodegradable surfactants are promising alternatives. For biosurfactants to be implemented industrially, they need to be produced on a large scale and also have tailorable properties that match those afforded by the polymerization of synthetic surfactants. In this paper, a scalable and versatile production method for biosurfactants based on a hydrophilic intrinsically disordered protein (IDP) sequence with a genetically engineered hydrophobic domain is used to study variables that impact their physicochemical and self-assembling properties. These amphiphilic sequences were found to self-assemble into micelles over a broad range of temperatures, pH values, and ionic strengths. To investigate the role of the IDP hydrophilic domain on self-assembly, variants with increased overall charges and systematically decreased IDP domain lengths were produced and examined for their sizes, morphologies, and critical micelle concentrations (CMCs). The results of these studies indicate that decreasing the length of the IDP domain and consequently the molecular weight and hydrophilic fraction leads to smaller micelles. In addition, significantly increasing the amount of charged residues in the hydrophilic IDP domain results in micelles of similar sizes but with higher CMC values. This represents an initial step in developing a quantitative model for the future engineering of biosurfactants based on this IDP sequence.

Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects.

Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote "longitudinal breathing" and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an "open hole" population on the order of 10-2 compared to 10-4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

Nucleotide and partner-protein control of bacterial replicative helicase structure and function.

Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α2ßß'ω·σA can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD.

Letting the cat out of the bag: a personal journey in Biophysics.

When the author arrived in Berkeley, in the mid 1970s, to study Biophysics he soon felt as if he was engaging himself in a somewhat marginal activity. Biology was then entering another of its cyclical periods of annotation that was to culminate with the human genome project. Two decades later, however, at the end of this process, it had become clear that two main tasks were acquiring a central importance in biological research: a renewed push for a quantitative, precise description of biological systems at the molecular level, and efforts towards an integrated understanding of the operation, control, and coordination of cellular processes. Today, these have become two of the most fertile research areas in Biophysics.

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism.

Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT's dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.

Limitations of constant-force-feedback experiments.

Single-molecule force spectroscopy has provided important insights into the properties and mechanisms of biological molecules and systems. A common experiment is to measure the force dependence of conformational changes at equilibrium. Here, we demonstrate that the commonly used technique of force feedback has severe limitations when used to evaluate rapid macromolecular conformational transitions. By comparing the force-dependent dynamics of three major classes of macromolecules (DNA, RNA, and protein) using both a constant-force-feedback and a constant-trap-position technique, we demonstrate a problem in force-feedback experiments. The finite response time of the instrument's force feedback can modify the behavior of the molecule, leading to errors in the reported parameters, such as the rate constants and the distance to the transition state, for the conformational transitions. We elucidate the causes of this problem and provide a simple test to identify and evaluate the magnitude of the effect. We recommend avoiding the use of constant force feedback as a method to study rapid conformational changes in macromolecules.

Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation.

Ribosomes are at the core of the central dogma of life. They perform the last major step of gene expression by translating the information written in the nucleotide codon sequences into the amino acid sequence of a protein. This is a complex mechanochemical process that requires the coordination of multiple dynamic events within the ribosome such as the precise timing of decoding and the subsequent translocation along the mRNA. We have previously used a high-resolution optical tweezers instrument with single-molecule fluorescence capabilities ("fleezers") to study how ribosomes couple binding of the GTPase translation elongation factor EF-G with internal conformational changes to unwind and progress across the mechanical barriers posed by mRNA secondary structures. Here, we present a detailed description of the procedures for monitoring two orthogonal channels (EF-G binding and translocation) by single actively translating ribosomes in real-time, to uncover the mechanism by which they harness chemical energy to generate mechanical force and displacement.

Optical tweezers in single-molecule biophysics.

Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein-nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth.

Pause sequences facilitate entry into long-lived paused states by reducing RNA polymerase transcription rates.

Transcription by RNA polymerase (RNAP) is interspersed with sequence-dependent pausing. The processes through which paused states are accessed and stabilized occur at spatiotemporal scales beyond the resolution of previous methods, and are poorly understood. Here, we combine high-resolution optical trapping with improved data analysis methods to investigate the formation of paused states at enhanced temporal resolution. We find that pause sites reduce the forward transcription rate of nearly all RNAP molecules, rather than just affecting the subset of molecules that enter long-lived pauses. We propose that the reduced rates at pause sites allow time for the elongation complex to undergo conformational changes required to enter long-lived pauses. We also find that backtracking occurs stepwise, with states backtracked by at most one base pair forming quickly, and further backtracking occurring slowly. Finally, we find that nascent RNA structures act as modulators that either enhance or attenuate pausing, depending on the sequence context.

Mechanisms of cellular proteostasis: insights from single-molecule approaches.

Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins.

Direct measurement of the mechanical work during translocation by the ribosome.

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano-chemical cycle.

Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism.

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation. DOI:http://dx.doi.org/10.7554/eLife.00971.001.