Dom34 Rescues Ribosomes in 3′ Untranslated Regions

SummaryRibosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3′ UTRs. These ribosomes appear to gain access to the 3′ UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them

Dwell time symmetry in random walks and molecular motors

The statistics of steps and dwell times in reversible molecular motors differ from those of cycle completion in enzyme kinetics. The reason is that a step is only one of several transitions in the mechanochemical cycle. As a result, theoretical results for cycle completion in enzyme kinetics do not apply to stepping data. To allow correct parameter estimation, and to guide data analysis and experiment design, a theoretical treatment is needed that takes this observation into account. In this paper, we model the distribution of dwell times and number of forward and backward steps using first passage processes, based on the assumption that forward and backward steps correspond to different directions of the same transition. We extend recent results for systems with a single cycle and consider the full dwell time distributions as well as models with multiple pathways, detectable substeps, and detachments. Our main results are a symmetry relation for the dwell time distributions in reversible motors, and a relation between certain relative step frequencies and the free energy per cycle. We demonstrate our results by analyzing recent stepping data for a bacterial flagellar motor, and discuss the implications for the efficiency and reversibility of the force-generating subunits. Key words: motor proteins; single molecule kinetics; enzyme kinetics; flagellar motor; Markov process; non-equilibrium fluctuations.Comment: revtex, 15 pages, 8 figures, 2 tables. v2: Minor revision, corrected typos, added references, and moved mathematical parts to new appendice

Kinesin Is an Evolutionarily Fine-Tuned Molecular Ratchet-and-Pawl Device of Decisively Locked Direction

Conventional kinesin is a dimeric motor protein that transports membranous organelles toward the plus-end of microtubules (MTs). Individual kinesin dimers show steadfast directionality and hundreds of consecutive steps, yetthe detailed physical mechanism remains unclear. Here we compute free energies for the entire dimer-MT system for all possible interacting configurations by taking full account of molecular details. Employing merely first principles and several measured binding and barrier energies, the system-level analysis reveals insurmountable energy gaps between configurations, asymmetric ground state caused by mechanically lifted configurational degeneracy, and forbidden transitions ensuring coordination between both motor domains for alternating catalysis. This wealth of physical effects converts a kinesin dimer into a molecular ratchet-and-pawl device, which determinedly locks the dimer's movement into the MT plus-end and ensures consecutive steps in hand-over-hand gait.Under a certain range of extreme loads, however, the ratchet-and-pawl device becomes defective but not entirely abolished to allow consecutive back-steps. This study yielded quantitative evidence that kinesin's multiple molecular properties have been evolutionarily adapted to fine-tune the ratchet-and-pawl device so as to ensure the motor's distinguished performance.Comment: 10 printed page

Nanoscale temperature measurements using non-equilibrium Brownian dynamics of a levitated nanosphere

Einstein realised that the fluctuations of a Brownian particle can be used to ascertain properties of its environment. A large number of experiments have since exploited the Brownian motion of colloidal particles for studies of dissipative processes, providing insight into soft matter physics, and leading to applications from energy harvesting to medical imaging. Here we use optically levitated nanospheres that are heated to investigate the non-equilibrium properties of the gas surrounding them. Analysing the sphere's Brownian motion allows us to determine the temperature of the centre-of-mass motion of the sphere, its surface temperature and the heated gas temperature in two spatial dimensions. We observe asymmetric heating of the sphere and gas, with temperatures reaching the melting point of the material. This method offers new opportunities for accurate temperature measurements with spatial resolution on the nanoscale, and a new means for testing non-equilibrium thermodynamicsComment: 5 pages, 4 figures, supplementary material available upon reques

Kinesin's backsteps under mechanical load

Kinesins move processively toward the plus end of microtubules by hydrolyzing ATP for each step. From an enzymatic perspective, the mechanism of mechanical motion coupled to the nucleotide chemistry is often well explained using a single-loop cyclic reaction. However, several difficulties arise in interpreting kinesin's backstepping within this framework, especially when external forces oppose the motion of kinesin. We review evidence, such as an ATP-independent stall force and a slower cycle time for backsteps, that has emerged to challenge the idea that kinesin backstepping is due to ATP synthesis, i.e., the reverse cycle of kinesin's forward-stepping chemomechanics. Supplementing the conventional single-loop chemomechanics with routes for ATP-hydrolyzing backward steps and nucleotide-free steps, especially under load, gives a better physical interpretation of the experimental data on backsteps.Comment: 5 figures and 2 table

Dying mRNA Tells a Story of Its Life

In this issue of Cell, Pelechano et al. report that sequencing of mRNA decay intermediates shows surprisingly tight coupling of a major decay pathway to the movement of the last translating ribosome, revealing stress- and starvation-dependent modulation of translation elongation

The relevance of neck linker docking in the motility of kinesin

Conventional kinesin is a motor protein, which is able to walk along a microtubule processively. The exact mechanism of the stepping motion and force generation of kinesin is still far from clear. In this paper we argue that neck linker docking is a crucial element of this mechanism, without which the experimentally observed dwell times of the steps could not be explained under a wide range of loading forces. We also show that the experimental data impose very strict constraints on the lengths of both the neck linker and its docking section, which are compatible with the known structure of kinesin.Comment: Accepted for publication in BioSystems as part of the proceedings of BIOCOMP 200

GTPBP1 resolves paused ribosomes to maintain neuronal homeostasis.

Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed i