Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme.

Massively parallel, quantitative measurements of biomolecular activity across sequence space can greatly expand our understanding of RNA sequence-function relationships. We report the development of an RNA-array assay to perform such measurements and its application to a model RNA: the core glmS ribozyme riboswitch, which performs a ligand-dependent self-cleavage reaction. We measure the cleavage rates for all possible single and double mutants of this ribozyme across a series of ligand concentrations, determining kcat and KM values for active variants. These systematic measurements suggest that evolutionary conservation in the consensus sequence is driven by maintenance of the cleavage rate. Analysis of double-mutant rates and associated mutational interactions produces a structural and functional mapping of the ribozyme sequence, revealing the catalytic consequences of specific tertiary interactions, and allowing us to infer structural rearrangements that permit certain sequence variants to maintain activity.

Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element.

Riboswitches modulate gene expression in response to small-molecule ligands. Switching is generally thought to occur via the stabilization of a specific RNA structure conferred by binding the cognate ligand. However, it is unclear whether any such stabilization occurs for riboswitches whose ligands also play functional roles, such as the glmS ribozyme riboswitch, which undergoes self-cleavage using its regulatory ligand, glucosamine 6-phosphate, as a catalytic cofactor. To address this question, it is necessary to determine both the conformational ensemble and its ligand dependence. We used optical tweezers to measure folding dynamics and cleavage rates for the core glmS ribozyme over a range of forces and ligand conditions. We found that the folding of a specific structural element, the P2.2 duplex, controls active-site formation and catalysis. However, the folded state is only weakly stable, regardless of cofactor concentration, supplying a clear exception to the ligand-based stabilization model of riboswitch function.

KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics.

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

Restoring Faculty Vitality in Academic Medicine When Burnout Threatens.

Increasing rates of burnout-with accompanying stress and lack of engagement-among faculty, residents, students, and practicing physicians have caused alarm in academic medicine. Central to the debate among academic medicine's stakeholders are oft-competing issues of social accountability; cost containment; effectiveness of academic medicine's institutions; faculty recruitment, retention, and satisfaction; increasing expectations for faculty; and mission-based productivity.The authors propose that understanding and fostering what contributes to faculty and institutional vitality is central to preventing burnout during times of change. They first look at faculty vitality and how it is threatened by burnout, to provide a framework for a greater understanding of faculty well-being. Then they draw on higher education literature to determine how vitality is defined in academic settings and what factors affect faculty vitality within the context of academic medicine. Next, they propose a model to explain and examine faculty vitality in academic medicine, followed by a discussion of the need for a greater understanding of faculty vitality. Finally, the authors offer conclusions and propose future directions to promote faculty vitality.The authors encourage institutional decision makers and other stakeholders to focus particular attention on the evolving expectations for faculty, the risk of extensive faculty burnout, and the opportunity to reduce burnout by improving the vitality and resilience of these talented and crucial contributors. Faculty vitality, as defined by the institution, has a critical role in ensuring future institutional successes and the capacity for faculty to thrive in a complex health care economy.

Real-time observation of polymerase-promoter contact remodeling during transcription initiation.

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the -35 and -10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (-10 to -35) and with the -10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

Intraflagellar transport velocity is governed by the number of active KIF17 and KIF3AB motors and their motility properties under load.

Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde intraflagellar transport (IFT), ferrying cargo along microtubules (MTs) toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by (i) the absolute numbers of each motor type on a given train, (ii) how prone KIF3AB is to dissociation from MTs relative to KIF17, and (iii) how prone both motors are to dissociation relative to binding MTs.

Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution.

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer.

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

Direct observation of processive exoribonuclease motion using optical tweezers.

Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.

Real-time observation of the initiation of RNA polymerase II transcription.

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase.

During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5' cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5' region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate-dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.

The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load.

The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1.

Examining kinesin processivity within a general gating framework.

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ~1 µm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.

Defining "faculty" in academic medicine: responding to the challenges of a changing environment.

Academic medicine in the United States is at a crossroads. There are many drivers behind this, including health care reform, decreased federal research funding, a refined understanding of adult learning, and the emergence of disruptive innovations in medicine, science, and education. As faculty members are at the core of all academic activities, the definition of "faculty" in academic medicine must align with the expectations of institutions engaged in patient care, research, and education. Faculty members' activities have changed and continue to evolve. Academic health centers must therefore define new rules of engagement that reflect the interplay of institutional priorities with the need to attract, retain, and reward faculty members. In this Commentary, the authors describe and explore the potential effects of the changing landscape for institutions and their clinical faculty members. The authors make a case for institutions to adapt faculty appointment, evaluation, and promotion processes, and they propose a framework for a standardized definition of "faculty" that allows for individual variability. This framework also provides a means to evaluate and reward faculty members' contributions in education, research, and clinical care. The authors propose a deliberate national conversation to ensure that careers in academic medicine remain attractive and sustainable and that the future of academic medicine is secure.

A DNA-based molecular probe for optically reporting cellular traction forces.

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

Kinesin processivity is gated by phosphate release.

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P(i)) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP · P(i) state (by adding free P(i)) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.

Reconstructing folding energy landscapes by single-molecule force spectroscopy.

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate.

A pause sequence enriched at translation start sites drives transcription dynamics in vivo.

Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms.

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.

Single-molecule studies of riboswitch folding.

The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.