细菌Hfq蛋白的结构、功能及作用机制

非编码小RNA(small RNA, sRNA)是细菌基因转录后调控的一个重要层次,也是近十年来原核生物研究领域的焦点之一。大多数sRNA的作用与Hfq蛋白密切相关,即Hfq可以促进sRNA与其靶标mRNA的互补配对,进而影响翻译的进行或者mRNA的稳定性。笔者对Hfq的结构、Hfq参与sRNA调节作用的机制、Hfq在多种细菌中的功能表型进行了综述。Hfq是一个保守的蛋白质,在很多细菌中广泛存在,并与真核生物中参与mRNA剪切与降解活动的Sm蛋白同源。在结构上,Hfq具有两个非等同的RNA结合面,可以结合并介导多个RNA分子的相互作用,其结构体现了和功能的高度统一性。目前,对Hfq的研究主要集中于革兰氏阴性细菌中,在革兰氏阳性细菌中,Hfq的功能尚不明晰; 此外,在许多重要的细菌中,Hfq影响功能表型的具体机制也不清楚。因此,今后有必要进一步精细研究Hfq的分子结构特征和功能特点,深入分析Hfq对细菌表型多样化的影响机制,探究Hfq影响靶标分子和功能表型的详尽机制。     

细菌非编码小RNA的生物学特征及作用机制研究进展

细菌非编码小RNA(sRNA)是一类长度为50～500 nt、一般不编码蛋白质的具有调控功能的小RNA,主要通过与靶mRNA碱基配对或与靶蛋白质结合,在转录后水平发挥调控作用.sRNA参与细菌氨基酸代谢和转运、环境胁迫响应以及毒力作用等众多重要生理过程的调控,与传统的转录因子等构成多层级的调控网络,协同作用使细菌对环境变化做出快速且精细的响应.由于sRNA在细菌调控网络中的重要作用,发现新的sRNA并阐明它们的调控功能成为原核生物基因表达调控研究的一个热点.本综述对细菌sRNA的筛选、鉴定、作用机制以及生物学功能等方面取得的进展进行总结,希望给相关领域的研究者提供一个较为全面的视角.     

非编码RNA调控心脏重构与再生

近年来, 越来越多的证据表明, 大量的非编码RNA(non-coding RNAs, ncRNAs)在基因的表达调控、细胞和机体的生理功能维持与病理环境调节方面都有重要作用, 其中主要包括微小RNA(microRNAs, miRNAs) 和长链非编码RNA(long non-coding RNAs, lncRNAs).心脏重构与再生是心血管疾病领域的关键问题, 其调控过程非常复杂, 包括表观遗传、转录、转录后及翻译水平的调控. 大量研究发现在转录后水平, miRNAs 通过负性调节靶标的表达调控心脏发育、疾病及再生进程. 近期研究揭示, lncRNAs 在心脏发育和疾病中具有潜在的作用, 可通过表观遗传、转录及转录后水平发挥作用. lncRNAs 已成为继miRNAs 之后的又一重要的调节性非编码RNA. 就非编码RNA 在心脏重构及再生进程中的调控作用进行综述.     

水生生物病原核酸适配体研究进展

核酸适配体(Aptamer)是指利用指数富集配体系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术筛选得到的能特异性识别和结合靶标的ssDNA或RNA分子,具有高亲和力和高特异性的特点,在快速检测和靶向治疗方面应用广阔。近年来,水生生物细菌和病毒性病原疾病的频繁暴发,严重制约了水生态的健康以及水产养殖业的迅速发展。核酸适配体作为一种新型的识别分子,在水生生物病原的应用上也已取得了一些进展。本文对水生生物细菌和病毒性病原核酸适配体的筛选,及其在检测和治疗领域的研究现状进行综述,并对该领域核酸适配体技术的应用方向进行展望。     

RNA切割型脱氧核酶在生物医学领域中的研究进展

RNA切割型脱氧核酶（RCD）是一类具有催化活性的单链DNA分子,具有灵活的可编程性、优异的稳定性、强底物特异性和高催化活性等优点,因而在生物医学领域展现出了良好的应用前景. 基于RCD对于特定辅助因子的依赖和对于靶标RNA的特异性识别和切割,其被广泛应用于一系列生物分子靶标的检测以及多种临床疾病的基因治疗. 文章介绍了RCD的物理化学特性,并对其在生物医学领域的研究现状进行了总结,聚焦于RCD在生物传感和癌症治疗等方面的应用进展. 此外,还对该领域面临的挑战及未来的发展方向进行了讨论.     

微RNA在非小细胞肺癌中的研究进展

微RNA(miRNA)是一类内源性、非编码的单链小分子RNA,作用广泛,参与生命活动中的一系列重要进程,并与肿瘤的发生、发展密切相关。大量研究表明,微RNA参与肺癌的发生、发展和预后,具有与原癌基因或肿瘤抑制基因相似的作用。在肺癌组织中微RNA有特定的表达谱,参与调节肿瘤血管生成等多个过程,并可作为生物标志物用以早期诊断,靶向治疗及临床预后。作为一类新的分子靶标,微RNA为肺癌的诊断和治疗提供了新的方向。     

伤寒沙门菌非编码RNA AsrC表达特性及功能初步研究

非编码RNA在细菌的各种生命活动中都发挥着重要的功能.针对伤寒沙门菌非编码RNA AsrC,对其转录和降解特性及其所具有的功能进行初步研究.首先使用Northern blot和qRT-PCR的方法检测了σ因子rpoE和rpoS对AsrC转录的影响,分析了使用利福平抑制细菌RNA合成的情况下,RNase E和RNase III对AsrC降解的影响.其次使用AsrC缺陷株和高表达菌株分析细菌在酸、氧、高渗应激条件下的生长情况.再者通过全基因组芯片技术,研究AsrC高表达菌株基因表达的变化.进一步研究AsrC在细菌侵袭和巨噬细胞胞内生存力中的作用.结果显示,rpoE在酸应激下、rpoS在高渗应激下调节AsrC的转录.细菌中RNase E主要参与了AsrC的降解.高表达AsrC后,有40个基因表达上调,23个基因表达下调.AsrC高表达可以增强伤寒沙门菌对于上皮细胞的侵袭力,并且可以减弱细菌的胞内生存和增殖力.     

铜绿假单胞菌毒力相关小RNA的筛选与鉴定

小RNA在细菌的基因调控过程中具有重要的作用,然而目前在铜绿假单胞菌中仅发现了几种有功能的小RNA.铜绿假单胞菌中存在着许多短片段RNA(100-250 bp),然而这些短片段RNA的功能尚不清楚.为了筛选与铜绿假单胞菌毒力相关的小RNA,结合生物信息学手段和实验检测技术对这些短片段RNA进行了靶点基因预测及相关表型研究.结果显示:利用小RNA靶点预测软件,筛选到23个毒力相关小RNA;通过构建小RNA过表达质粒,对这些小RNA进行了高表达及相关的表型检测,发现Pant116对铜绿假单胞菌的3型分泌系统具有负调控作用.     

细菌纤维素的生物合成机理、理化性质及应用研究进展

细菌纤维素是一种以革兰氏阴性细菌为主的多个菌属合成的天然有机高分子材料,与植物纤维素化学结构相同.微生物以葡萄糖为底物分子,通过多酶系系统合成的细菌纤维素纯度高、结晶度高、吸水能力强、稳定性好、生物安全性突出,目前已被广泛应用于食品、医药、电化学、环境修复等多个领域.综述了细菌纤维素的合成过程、理化性质及应用研究进展,并展望了未来研究的主要方向.     

一种简便高效提取细菌总DNA和RNA的方法

本科实验教学中,细菌总DNA和RNA的提取是一个基础实验.但该实验中经常出现DNA提取不出来、产量低、条带不清晰、RNA降解、蛋白残留多等问题,影响了学生对细菌DNA和RNA的直观认识,降低了实验教学效果.将常规提取方法进行精简优化,摸索出一种简便、稳定、高效、成功率高的DNA和RNA同时提取的方法.所获样品电泳条带清晰,无降解,蛋白残留少;且实验步骤少,基本每个学生都能获得理想结果,明显改善了教学实验效果,值得在本科生物化学实验教学中推广.     

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.     

Pervasive roles of microRNAs in cardiovascular biology

First recognized as regulators of development in worms and fruitflies, microRNAs are emerging as pivotal modulators of mammalian cardiovascular development and disease. Individual microRNAs modulate the expression of collections of messenger RNA targets that often have related functions, thereby governing complex biological processes. The wideranging functions of microRNAs in the cardiovascular system have provided new perspectives on disease mechanisms and have revealed intriguing therapeutic targets, as well as diagnostics, for a variety of cardiovascular disorders.     

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.     

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.     

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.     

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the world's largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific 'ladderane' membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.     

Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor

We show that nerve growth factor (NGF) synthesis in developing skin begins with sensory innervation and that sensory neurons do not express NGF receptors until their fibres reach their cutaneous targets. Both cutaneous epithelium and mesenchyme synthesize NGF, the concentration of messenger RNA for NGF being higher in the more densely innervated epithelium.