Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.     

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.     

作用于cip-cel mRNA的核糖核酸内切酶的鉴定

【目的】本研究以革兰氏阳性细菌解纤维素梭菌(Ruminiclostridium cellulolyticum)为研究对象,筛选作用于纤维小体编码基因簇cip-cel mRNA的核糖核酸内切酶。【方法】通过对预测的4个假定编码核糖核酸内切酶基因进行基因敲除、体内过表达、体外过表达和活性分析等方法,分析它们对cip-cel mRNA剪切位点的剪切能力。【结果】敲除rnc和rnj基因,对cip-cel mRNA剪切没有任何影响;体内过表达RNase时能够加速cip-cel mRNA的降解,而过表达RNase G时,则结果与野生型对照菌株无异;RNase G基因rng和RNase Y基因rny的体外活性鉴定分析,发现RNase G对体外转录的包含cip-cel mRNA剪切位点的RNA没有作用,而RNase Y能够对其进行剪切和降解。【结论】RNase Y是能够作用于cip-cel mRNA的核酸内切酶。该研究结果对理解革兰氏阳性细菌核糖核酸内切酶的作用机制,及其在转录后水平的调控基因差异表达等方面具有重大意义。     

RNase J1 endonuclease activity as a probe of RNA secondary structure

Reliable determination of RNA secondary structure depends on both computer algorithms and experimental probing of nucleotides in single- or double-stranded conformation. Here we describe the exploitation of the endonucleolytic activity of the Bacillus subtilis enzyme RNase J1 as a probe of RNA structure. RNase J1 cleaves in single-stranded regions and, in vitro at least, the enzyme has relatively relaxed nucleotide specificity. We confirmed the feasibility of the approach on an RNA of known structure, B. subtilis tRNA^Thr. We then used RNase J1 to solve the secondary structure of the 5′ end of the hbs mRNA. Finally, we showed that RNase J1 can also be used in footprinting experiments by probing the interaction between the 30S ribosomal subunit and the Shine–Dalgarno element of the hbs mRNA.