Conformational dynamics of the RNA binding channel regulates loading and translocation of the DEAH-box helicase Prp43.

The DEAH-box helicase Prp43 has essential functions in pre-mRNA splicing and ribosome biogenesis, remodeling structured RNAs. To initiate unwinding, Prp43 must first accommodate a single-stranded RNA segment into its RNA binding channel. This allows translocation of the helicase on the RNA. G-patch (gp) factors activate Prp43 in its cellular context enhancing the intrinsically low ATPase and RNA unwinding activity. It is unclear how the RNA loading process is accomplished by Prp43 and how it is regulated by its substrates, ATP and RNA, and the G-patch partners. We developed single-molecule (sm) FRET reporters on Prp43 from Chaetomium thermophilum to monitor the conformational dynamics of the RNA binding channel in Prp43 in real-time. We show that the channel can alternate between open and closed conformations. Binding of Pfa1(gp) and ATP shifts the distribution of states towards channel opening, facilitating the accommodation of RNA. After completion of the loading process, the channel remains firmly closed during successive cycles of ATP hydrolysis, ensuring stable interaction with the RNA and processive translocation. Without Pfa1(gp), it remains predominantly closed preventing efficient RNA loading. Our data reveal how the ligands of Prp43 regulate the structural dynamics of the RNA binding channel controlling the initial binding of RNA.

Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1.

The DEAH/RHA helicase Prp43 remodels protein-RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are intrinsically low and become activated by G-patch (gp) factors in the specific cellular context. The gp motif connects the helicase core to the flexible C-terminal domains, but it is unclear how this affects RecA domain movement during catalysis and the unwinding of RNA substrates. We developed single-molecule Förster Resonance Energy Transfer (smFRET) reporters to study RecA domain movements within Prp43 in real time. Without Pfa1(gp), the domains approach each other adopting predominantly a closed conformation. The addition of Pfa1(gp) induces an open state, which becomes even more prevalent during interaction with RNA. In the open state, Prp43 has reduced contacts with bound nucleotide and shows rapid adenosine diphosphate (ADP) release accelerating the transition from the weak (ADP) to the strong (apo) RNA binding state. Using smFRET labels on the RNA to probe substrate binding and unwinding, we demonstrate that Pfa1(gp) enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states instead of dissociating from the RNA. ATP binding to the apo-enzyme induces the translocation along the RNA, generating the unwinding force required to melt proximal RNA structures. During ATP turnover, Pfa1(gp) stimulates alternating of the RecA domains between open and closed states. Consequently, the translocation becomes faster than dissociation from the substrate in the ADP state, allowing processive movement along the RNA. We provide a mechanistic model of DEAH/RHA helicase motility and reveal the principles of Prp43 regulation by G-patch proteins.

Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting.

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and -1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the -1-frame triplet, resulting in slippage followed by recruitment of the -1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Dynamics of ribosomes and release factors during translation termination in E. coli.

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

Kinetics of Spontaneous and EF-G-Accelerated Rotation of Ribosomal Subunits.

Ribosome dynamics play an important role in translation. The rotation of the ribosomal subunits relative to one another is essential for tRNA-mRNA translocation. An important unresolved question is whether subunit rotation limits the rate of translocation. Here, we monitor subunit rotation relative to peptide bond formation and translocation using ensemble kinetics and single-molecule FRET. We observe that spontaneous forward subunit rotation occurs at a rate of 40 s(-1), independent of the rate of preceding peptide bond formation. Elongation factor G (EF-G) accelerates forward subunit rotation to 200 s(-1). tRNA-mRNA movement is much slower (10-40 s(-1)), suggesting that forward subunit rotation does not limit the rate of translocation. The transition back to the non-rotated state of the ribosome kinetically coincides with tRNA-mRNA movement. Thus, large-scale movements of the ribosome are intrinsically rapid and gated by its ligands such as EF-G and tRNA.

Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome.

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

Properties of the kinesin-3 NcKin3 motor domain and implications for neck function.

Members of the Kinesin-3 family are microtubule motors involved in the transport of membranous cargo. NcKin3 from the fungus Neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. To determine how one of the heads is inactivated, we investigated truncated monomeric constructs. None of the constructs generated processive single-molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. The kinetic analysis suggests futile ATP hydrolysis cycles, because a representative monomer showed a faster ATP turnover than the dimer while supporting slower motility. The K(0.5,MT) was 70-fold lower, the microtubule-bound portion of the kinetic cycle eight-fold longer and the microtubule detachment rate almost 15-fold slower than that of the dimer. Moreover, the monomer's microtubule-dependent ADP release occurred three-fold to four-fold faster than k(cat) (125 versus 34 s(-1)), whereas phosphate release was approximately equally fast (29 s(-1)). A dimeric construct containing a structure-breaking insert between motor head and neck showed a similar behaviour. These data suggest that the heads of the wild-type NcKin3 motor are strictly coupled via the neck domain, and that the dimeric structure is required for proper detachment after one ATPase cycle. This is the first direct comparison of a monomeric Kinesin-3 with its dimeric full-length counterpart, and the kinetic changes observed here may also apply to other Kinesin-3 motors.

Dissection of kinesin's processivity.

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil.

The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression.

BACKGROUND: The accurate alignment of chromosomes at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic integrity of eukaryotes. Although it is well established that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well understood. RESULTS: By combining RNAi-depletion experiments with in vitro biochemical assays, we demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells. We show that in vitro Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A like its yeast ortholog Kip3p depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies revealed that in Kif18A-depleted cells, chromosomes move at reduced speed and completely fail to align at the spindle equator. CONCLUSIONS: These studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.

Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3.

Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.

Peptide bond formation does not involve acid-base catalysis by ribosomal residues.

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).

Review: regulation mechanisms of Kinesin-1.

Kinesin-1 microtubule motors are common kinesin motors from protozoa, fungi and animals. They transport vesicular or particle cargo in a strictly regulated manner. The relatively well-studied tail inhibition mechanism is based on a conformational change that leads to an interaction of Kinesin-1's tail with the junction of neck and hinge regions. This folding causes a decrease in microtubule binding and motor activity. In fungal Kinesin-1 motors several lines of evidence suggest that a conserved tyrosine in the neck coiled-coil mediates this inhibition. In the active state, a region surrounding a conserved tryptophan in the hinge stabilises the neck coiled-coil, and prevents the tyrosine from inhibiting. Although animal and fungal Kinesin-1 motors are clearly homologous and function according to the same chemo-mechanical mechanism, they differ in their regulation. Unlike fungal Kinesin-1s, animal kinesins associate with light chains that are important for regulation and cargo interaction. Several proteins interacting with animal Kinesin-1 heavy or light chains are known, among them typical scaffolding proteins that seem to link Kinesin-1 to signalling pathways.

The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation.

Peptide bond formation on the ribosome is catalyzed by RNA. Kinetic studies using Escherichia coli ribosomes have shown that catalysis (>10(5)-fold overall acceleration) is due to a large part to substrate positioning. However, peptide bond formation is inhibited approximately 100-fold by protonation of a ribosomal group with pKa=7.5, indicating either a contribution of general acid-base catalysis or inhibition by a pH-dependent conformational change within the active site. The function of a general base has been attributed to A2451 of 23S rRNA, and a charge relay system involving G2447 has been postulated to bring about the extensive pKa shift of A2451 implied in the model. Using a rapid kinetic assay, we found that the G2447A mutation, which has essentially no effect on cell growth, lowers the rate of peptide bond formation about 10-fold and does not affect the ionization of the ribosomal group with pKa=7.5 taking part in the reaction. This result does not support the proposed charge relay mechanism involving G2447 and the role of A2451 as general base in the catalysis of peptide bond formation.