Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

<div><p>SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed <i>in vitro</i> translation assays with HaloTag proteins fused to the C-terminal fragment of <i>E</i>. <i>coli</i> SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest.</p></div

In vitro translation of HaloTag proteins with mutated arrest sequence.

<p>Each protein construct, with or without a mutation (R163A or P166A) in the arrest sequence, was translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture incubated at 37°C for 3 min. Aliquots were withdrawn before and 3 min after the addition of puromycin and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. <b>A</b>, Halo-L8-SecM_133–170; <b>B</b>, Halo-L17-SecM_133–170; <b>C</b>, Halo-L26-SecM_133–170; <b>D</b>, Halo-pD-L8-SecM_133–170; <b>E</b>, Halo-SecM_1–170. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results.</p

Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence.

<p>(<b>A</b>-<b>D</b>) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM_133–170 (<b>A</b>), Halo-L26-SecM_133–170 (<b>B</b>), Halo-pD-L8-SecM_133–170 (<b>C</b>) and Halo-SecM_1–170 (<b>D</b>) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. (<b>E</b>) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM_133–170; diamonds, Halo-L26-SecM_133–170; triangles, Halo-pD-L8-SecM_133–170; circles, Halo-SecM_1–170. Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM_133–170, Halo-L26-SecM_133–170, Halo-pD-L8-SecM_133–170 and Halo-SecM_1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). (<b>F</b>) Time-course analysis of myc-SecM_1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM_1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM_1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. (<b>G</b>) The fraction of myc-SecM_1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM_1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).</p

In vitro translation of HaloTag proteins harbouring the arrest sequence.

<p>(<b>A</b>) Halo-L8-SecM_133–170 (lane 1), Halo-L17-SecM_133–170 (lane 2), Halo-L26-SecM_133–170 (lane 3), Halo-pD-L8-SecM_133–170 (lane 4) and Halo-SecM_1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. (<b>B</b>) Myc-Halo-L8-SecM_133–170 (lane 1), myc-Halo-L17-SecM_133–170 (lane 2), myc-Halo-L26-SecM_133–170 (lane 3), myc-Halo-pD-L8-SecM_133–170 (lane 4) and myc-Halo-SecM_1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. (<b>C</b>) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's <i>t</i>-test (<i>p</i> < 0.05).</p

Synthesis of a Pillar[5]arene-Based [2]Rotaxane with Two Equivalent Stations via Copper(I)-Catalyzed Alkyne–Azide Cycloaddition

A one-pot synthesis of pillar[5]­arene-based [2]­rotaxanes containing one and two stations by copper­(I)-catalyzed alkyne–azide cycloaddition (CuAAC) reaction is reported. In situ formation of the two stations by two stepwise CuAAC reactions allows for the synthesis of a [2]­rotaxane containing two stations with equal energy levels that exhibit shuttling of the pillar[5]­arene wheel

Cyclic Host Liquids for Facile and High-Yield Synthesis of [2]Rotaxanes

We developed “cyclic host liquids (CHLs)” as a new type of solvent. The CHLs are a nonvolatile liquid over a wide temperature range, are biocompatible and recyclable, have high thermal stability, and are miscible with many organic solvents. Compared with typical complexation systems, the CHL system is extremely efficient for maintaining host–guest complexation because an additional solvent is not required. Based on the efficient host–guest complexation in the CHL system, we demonstrated synthesis of [2]­rotaxanes in pillar[5]­arene-based CHL. High yields were obtained for [2]­rotaxanes capped by cationization (yield 91%) and Huisgen reaction (yield 88%) between the axle and the stopper components in the CHL system, while the association constants between the axles and wheels were quite low (10–15 M^–1) in CDCl₃. The CHL system provides a new powerful approach for synthesis of mechanically interlocked molecules (MIMs) even with unfavorable statistical combinations of host–guest complexes

Data File 1: Atomic scattering factor of the ASTRO-H (Hitomi) SXT reflector around the gold’s L edges

Atomic Scattering Factors Originally published in Optics Express on 31 October 2016 (oe-24-22-25548