Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94.

Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.

Alternative transcription cycle for bacterial RNA polymerase.

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone.

The Hsp90 family of chaperones requires ATP-driven cycling to perform their function. The presence of two bound ATP molecules is known to favor a closed conformation of the Hsp90 dimer. However, the structural and mechanistic consequences of subsequent ATP hydrolysis are poorly understood. Using single-molecule FRET, we discover novel dynamic behavior in the closed state of Grp94, the Hsp90 family member resident in the endoplasmic reticulum. Under ATP turnover conditions, Grp94 populates two distinct closed states, a relatively static ATP/ATP closed state that adopts one conformation, and a dynamic ATP/ADP closed state that can adopt two conformations. We constructed a Grp94 heterodimer with one arm that is catalytically dead, to extend the lifetime of the ATP/ADP state by preventing hydrolysis of the second ATP. This construct shows prolonged periods of cycling between two closed conformations. Our results enable a quantitative description of how ATP hydrolysis influences Grp94, where sequential ATP hydrolysis steps allow Grp94 to transition between closed states with different dynamic and structural properties. This stepwise transitioning of Grp94's dynamic properties may provide a mechanism to propagate structural changes to a bound client protein.

Synergistic assembly of human pre-spliceosomes across introns and exons.

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation.

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging.

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.

Ordered and dynamic assembly of single spliceosomes.

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

Processive movement by a kinesin heterodimer with an inactivating mutation in one head.

A single molecule of the motor enzyme kinesin-1 keeps a tight grip on its microtubule track, making tens or hundreds of discrete, unidirectional 8 nm steps before dissociating. This high duty ratio processive movement is thought to require a mechanism in which alternating stepping of the two head domains of the kinesin dimer is driven by alternating, overlapped cycles of ATP hydrolysis by the two heads. The R210K point mutation in Drosophila kinesin heavy chain was reported to disrupt the ability of the enzyme active site to catalyze ATP P-O bond cleavage. We expressed R210K homodimers as well as isolated R210K heads and confirmed that both are essentially inactive. We then coexpressed tagged R210K subunits with untagged wild-type subunits and affinity purified R210K/wild-type heterodimers together with the inactive R210K homodimers. In contrast to the R210K head or homodimer, the heterodimer was a highly active (>50% of wild-type) microtubule-stimulated ATPase, and the heterodimer displayed high duty ratio processive movement in single-molecule motility experiments. Thus, dimerization of a subunit containing the inactivating mutation with a functional subunit can complement the mutation; this must occur either by lowering or by bypassing kinetic barriers in the ATPase or mechanical cycles of the mutant head. The observations provide support for kinesin-1 gating mechanisms in which one head stimulates the rate of essential processes in the other.

Two distinct modes of processive kinesin movement in mixtures of ATP and AMP-PNP.

An enzyme is frequently conceived of as having a single functional mechanism. This is particularly true for motor enzymes, where the necessity for tight coupling of mechanical and chemical cycles imposes rigid constraints on the reaction pathway. In mixtures of substrate (ATP) and an inhibitor (adenosine 5'-(beta,gamma-imido)triphosphate or AMP-PNP), single kinesin molecules move on microtubules in two distinct types of multiple-turnover "runs" that differ in their susceptibility to inhibition. Longer (less susceptible) runs are consistent with movement driven by the alternating-sites mechanism previously proposed for uninhibited kinesin. In contrast, kinesin molecules in shorter runs step with AMP-PNP continuously bound to one of the two active sites of the enzyme. Thus, in this mixture of substrate and inhibitor, kinesin can function as a motor enzyme using either of two distinct mechanisms. In one of these, the enzyme can accomplish high-duty-ratio processive movement without alternating-sites ATP hydrolysis.

Imaging of single-molecule translocation through nuclear pore complexes.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements.

The motor enzyme kinesin makes hundreds of unidirectional 8-nanometer steps without detaching from or freely sliding along the microtubule on which it moves. We investigated the kinesin stepping mechanism by immobilizing a Drosophila kinesin derivative through the carboxyl-terminal end of the neck coiled-coil domain and measuring orientations of microtubules moved by single enzyme molecules at submicromolar adenosine triphosphate concentrations. The kinesin-mediated microtubule-surface linkage was sufficiently torsionally stiff (>/=2.0 +/- 0.9 x 10(-20) Newton meters per radian2) that stepping by the hypothesized symmetric hand-over-hand mechanism would produce 180 degree rotations of the microtubule relative to the immobilized kinesin neck. In fact, there were no rotations, a finding that is inconsistent with symmetric hand-over-hand movement. An alternative "inchworm" mechanism is consistent with our experimental results.