The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription

Shingo Fukuda, Shannon Yan, Yusuke Komi, Mingxuan Sun, Ronen Gabizon, Carlos Bustamante

The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5′-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.

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Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by Structured mRNAs

Varsha P. Desai, Filipp Frank, Antony Lee, Maurizio Righini, Laura Lancaster, Harry F.Noller, Ignacio Tinoco Jr., Carlos Bustamante

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

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The Ribosome Cooperates with a Chaperone to Guide Multi-domain Protein Folding

Kaixian Liu, Kevin Maciuba, Christian M.Kaiser

Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.

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Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection

Cedric R. Clapier, Margaret M. Kasten, Timothy J. Parnell, Ramya Viswanathan, Heather Szerlong, George Sirinakis, Yongli Zhang, Bradley R. Cairns

The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving “coupling”—the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote “coupling,” and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.

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Mechanism of Homology Recognition in DNA Recombination from Dual-Molecule Experiments

Iwijn De Vlaminck, Marijn T.J. van Loenhout, Ludovit Zweifel, Johan den Blanken, Koen Hooning, Susanne Hage, Jacob Kerssemakers, Cees Dekker

In E. coli homologous recombination, a filament of RecA protein formed on DNA searches and pairs a homologous sequence within a second DNA molecule with remarkable speed and fidelity. Here, we directly probe the strength of the two-molecule interactions involved in homology search and recognition using dual-molecule manipulation, combining magnetic and optical tweezers. We find that the filament's secondary DNA-binding site interacts with a single strand of the incoming double-stranded DNA during homology sampling. Recognition requires opening of the helix and is strongly promoted by unwinding torsional stress. Recognition is achieved upon binding of both strands of the incoming dsDNA to each of two ssDNA-binding sites in the filament. The data indicate a physical picture for homology recognition in which the fidelity of the search process is governed by the distance between the DNA-binding sites.

DOI

Torque Generation of Kinesin Motors Is Governed by the Stability of the Neck Domain

Melanie Brunnbauer, Renate Dombi, Thi-Hieu Ho, Manfred Schliwa, Matthias Rief, Zeynep Ökten

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules. In stark contrast to kinesin-1, these motors display a wide range of left-handed spiraling around microtubules and thus generate torque during cargo transport. We provide direct evidence that kinesin's neck region determines the torque-generating properties. A model system based on kinesin-1 corroborates this result: disrupting the stability of the neck by inserting flexible peptide stretches resulted in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduced the spiraling of the motor up to the point of protofilament tracking. Finally, we present a model that explains the physical basis of kinesin's spiraling around the microtubule. 

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Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA

Jing Zhou, Kook Sun Ha, Arthur La Porta, Robert Landick, Steven M. Block

Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ∼19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.

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