The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

The scavenger decapping enzyme hydrolyses the protecting 5′ cap structure from short mRNAs that result from exosomal degradation. Based on static crystal structures and NMR data it is apparent that the dimeric enzyme has to undergo large structural changes to bind substrate in a catalytically competent conformation. Here, we study the yeast enzyme and show that the associated opening-closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by binding of a second ligand to the enzyme, which occurs under high substrate concentrations. We designed a mutant that disrupts the allosteric pathway that links the second binding event to the dynamics and show that this mutant enzyme is hyperactive. Our data reveals a unique mechanism of substrate inhibition, where motions that are required for catalytic activity also inhibit efficient turnover, when they are present in excess.

Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5′→3′ exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information.

The 5′ messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3′ end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3′ to 5′ degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.

<p>For the design of mitigation measures knowledge of debris-flow impact forces, usually estimated based on hydrostatic, hydrodynamic, or combined approaches, is essential. As these approaches are based on Newtonian fluids, they must be adjusted by empirical correction factors to account for the solid-fluid nature of debris flows. The values for the correction factors shown in the literature vary over a wide range and several studies showed a clear dependence with the Froude regime of debris flows.</p><p>To better understand the correction factors and to be able to calculate them using parameters that describe the flow behaviour a total of 32 experiments were conducted in the course of the project “Debris flow impact forces on bridge super structures (DEFSUP)”, funded by the Austrian Science Fund (FWF). Two different material compositions, different water contents as well as a total impact and a bypassing of the measuring block were tested.</p><p>The experimental setup designed within the project consists of a 4 m long semi-circular channel with a diameter of 300 mm and an inclination of 20°. The material is released from a rectangular reservoir in a dam-break scenario and accelerated with zero roughness on a length of 1.2 m and transferred to the semi-circle profile. The subsequently introduced roughness with a grain diameter of 1-2 mm generates a stationary phenomenological debris flow until it hits the measuring setup. With a starting volume of 50 kg, flow heights between 8 and 12 cm and velocities from 0.8 to 2.2 m/s were achieved according to the material composition and different water content. With these different mixtures a Froude-range from 0.6 to 3.6 was covered. In addition, normal stresses and pore water pressures were measured at the exact same point.</p><p>A detailed analysis of the measured impact forces together with the above mentioned measured parameters showed that the hydrodynamic correction factor is a constant mainly corresponding to the liquification ratio of the debris-flow mixture. Hence, the hydrodynamic correction factor can be regarded as a drag coefficient and seems to depend mainly on the internal friction of the flowing medium. At low Froude numbers measured impact forces exceed even a full momentum transfer if the mean bulk density is used for the calculation. This indicates that the impact forces can no longer be described by the hydrodynamic approach alone. For this reason, an additional pressure term based on a hydrostatic approach is considered in the combined concept. This additional pressure term depends on the dynamics of flow (Froude number) and can be modelled via a dynamic earth pressure coefficient.</p><p>The findings from these experiments contribute to a better prediction of debris-flows impact forces in terms of their material composition and flow behaviour.</p>

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