Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli.

Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.     

流加培养重组大肠杆菌表达核糖核酸酶Ba

核糖核酸酶Ba(barnase)是一种产自解淀粉芽孢杆菌的胞外核糖核酸酶,具有强毒性,能降解RNA,在农业、医疗、制药等领域有广泛的应用前景.利用重组表达barnase的大肠杆菌为研究对象,以期使用kil基因改善分泌状况,并实现大肠杆菌的高密度培养以及蛋白的高产表达.通过把kil-Km分泌盒克隆入质粒载体pET-32a(+)-barnase-barstar使barnase更加容易分泌与纯化,并使用Riesenberg培养基对重组大肠杆菌发酵生产barnase的不同葡萄糖浓度底物反馈控制补料流加策略进行了研究.初步确定1.0 g/L葡萄糖底物反馈流加时,获得的barnase酶活最高,为7.14 kU/mL,是文献报道的1.88倍,此时细胞干质量浓度为22.93 g/L,其中细胞间质的酶活达到3.53 kU/mL.为今后的发酵bar-nase研究提供了参考.     

Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.     

Structure and conserved RNA binding of the PAZ domain

The discovery of RNA-mediated gene-silencing pathways, including RNA interference, highlights a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defence. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. Notably, these two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown. Here we report the nuclear magnetic resonance solution structure of the PAZ domain from Drosophila melanogaster Argonaute 1 (Ago1). The structure consists of a left-handed, six-stranded beta-barrel capped at one end by two alpha-helices and wrapped on one side by a distinctive appendage, which comprises a long beta-hairpin and a short alpha-helix. Using structural and biochemical analyses, we demonstrate that the PAZ domain binds a 5-nucleotide RNA with 1:1 stoichiometry. We map the RNA-binding surface to the open face of the beta-barrel, which contains amino acids conserved within the PAZ domain family, and we define the 5'-to-3' orientation of single-stranded RNA bound within that site. Furthermore, we show that PAZ domains from different human Argonaute proteins also bind RNA, establishing a conserved function for this domain.     

核糖核酸酶Ⅲ超家族与RNA干扰

核糖核酸酶Ⅲ(RNaseⅢ)是一个在生物体中普遍存在的酶类,它具有把RNA前体加工成有功能的RNA、参与RNA干扰和其它细胞活性的功能.RNaseⅢ超家族包括大肠杆菌的核糖核酸酶Ⅲ(RNaseⅢ)、酵母核糖核酸酶t1(Rnt1)、植物中的核糖核酸酶Dicer和哺乳动物中的核糖核酸酶Drosha,它们都具有在特定位点或者特定的序列区域识别和切割双链核糖核酸(dsRNA)的能力.对RNaseⅢ超家族的种类、结构和功能、作用机制及其在RNA干扰中所起的重要作用以及在生命科学研究中的应用进行了归纳总结.     

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.     

Structure of the SRP19 RNA complex and implications for signal recognition particle assembly

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.     

构建的FIt3L胞外域原核表达载体在大肠杆菌中的表达

目的:构建含组氨酸标签的FMS样酪氨酸激酶3配体(Fms-like tyrosine kinase 3 ligand,FIt3L)胞外域蛋白的原核表达载体,并在大肠杆菌中进行表达.方法:依据FIt3L基因序列设计引物,以RT-PCR的方法从小鼠脾脏总RNA中克隆FIt3L基因并构建其胞外域原核表达载体,测序鉴定后在大肠杆菌BL21(DE3)菌株中诱导表达,用免疫印迹鉴定.结果:克隆到FIt3L编码区全长序列,经DNA测序证明与已报道的序列相同;构建羧基端带有His6标签的FIt3L胞外域蛋白的原核表达载体,转化大肠杆菌后SDS-PAGE分析显示在BL21(ED3)中获得较高表达;免疫印迹分析表明IPTG诱导后表达的FIt3L胞外域蛋白的相对分子质量(Mr)为19 000,与理论值相符,此蛋白与抗His6标签的抗体发生特异性反应.结论:成功克隆FIt3L基因,构建其胞外域蛋白的原核表达载体,并在大肠杆菌中获得表达.