Finding Protein Sequence Motifs--methods And Applications

基因组序列和蛋白质结构在过去十年中的快速积累已经被序列数据库搜索方法的重大进展所证实。NCBI开发的强大的位置特异性迭代BLAST（PSI-BLAST）方法构成了我们蛋白质基序分析工作的基础。此外，隐马尔可夫模型（HMM），在HHSearch方法中实现的蛋白质轮廓对轮廓比较，蛋白质结构比较方法和基因组上下文分析被广泛应用。 在过去的一年里，我们在详细分析几类蛋白质和结构域的分类，进化和功能方面取得了进一步的进展。具体而言，我们详细研究了原核生物中参与获得性免疫的蛋白质复合物和功能系统。存在于大多数细菌和许多细菌中的CRISPR-Cas适应性免疫系统通过将外来基因组的片段掺入特定的基因组基因座中，转录插入物并使用转录物作为指导RNA来破坏同源病毒或质粒的基因组来发挥作用。这种RNA干扰样免疫应答由许多不同且快速进化的Cas（CRISPR相关）蛋白介导，其中几种形成Cascade复合物，参与CRISPR转录物的加工和靶DNA的切割。我们对Cas蛋白序列和结构的比较分析导致CRISPR-Cas系统被新分类为三种不同的类型（I，II和III）。Cas蛋白的可用序列和结构的详细比较揭示了几个以前未被注意的同源关系。包含不同形式的RNA识别基序（RRM）结构域的重复相关神秘蛋白（RAMP）是CRISPR-Cas系统的主要组成部分，被分为三大组：Cas 5，Cas 6和Cas 7。这些组中的每一个包括许多以前未表征的蛋白质，现在显示采用RAMP结构。有证据表明，大多数CRISPR-Cas系统中含有的大亚基可能与Cas 10蛋白同源，Cas 10蛋白含有聚合酶样Palm结构域，并且预计在III型CRISPR-Cas系统中具有酶活性，但在I型系统中失活。这些发现，CRISPR聚合酶、RAMP和Cas 2都含有核心RRM结构域的事实，以及三种类型的CRISPR-Cas系统中不同的基因排列，共同为CRISPR-Cas机制的起源和进化提供了一个简单的场景。在这种情况下，CRISPR-Cas系统起源于嗜热菌，随后在原核生物中水平传播。 因此。以前认为不相关的Cas蛋白家族的统一提供了CRISPR-Cas系统分类的实质性改进和它们进化的重建。 此外，我们已经广泛地表征了蛋白质结构域，并预测了许多新的蛋白质复合物参与原核生物中其他形式的抗病毒防御。细胞生命形式和病毒之间的军备竞赛是进化的主要驱动力。细菌和古细菌基因组的很大一部分致力于抗病毒防御。我们分析了细菌和古细菌基因组中防御基因和典型的移动组成分（如病毒和转座子基因）的分布，并证明了抗病毒防御系统和移动的基因和元件在基因组岛中的统计学显著聚类。防御岛富含推定的操纵子，并包含许多过度代表的基因家族。对这些家族中基因编码的蛋白质的详细序列分析表明，它们中的许多是已知防御系统组分的分歧变体，而其他的则显示出特征，例如特征性操纵子组织，这暗示了新的防御系统。因此，基因组岛为细菌和古细菌抗病毒防御的实验研究提供了丰富的材料。除了CRISPR-Cas系统之外，不同类别的防御系统，特别是毒素-抗毒素和限制-修饰系统，在防御岛中显示出非随机聚类。目前尚不清楚这些协会反映了不同防御系统之间的功能合作，以及这些岛屿在多大程度上是积累各种非必需基因的基因组“汇”，特别是那些通过HGT获得的基因。防御岛的特性类似于机动岛。防御和mobilome基因是非随机关联的岛屿，这表明非适应性进化的岛屿通过一个优惠的附件样机制的防御系统，如毒素-抗毒素和水平移动性在这些岛屿的进化中的重要作用的成瘾性的基础上。

The rapid accumulation of genome sequences and protein structures during the last decade has been paralleled by major advances in sequence database search methods. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI formed the basis of our work on protein motif analysis. In addition, Hidden Markov Models (HMM), protein profile-against-profile comparison implemented in the HHSearch method, protein structure comparison methods and genome context analysis were extensively applied. Over the last year, we made further progress in detailed analysis of the classification, evolution, and functions of several classes of proteins and domains. Specifically, we studied in detail the protein complexes and functional systems that are involved in adaptive immunity in prokaryotes. The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Our comparative analysis of the Cas protein sequences and structures led to a new classification of the CRISPR-Cas systems into three distinct Types (I, II and III). A detailed comparison of the available sequences and structures of Cas proteins revealed several previously unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Thus. unification of Cas protein families previously considered unrelated provided for substantial improvement in the classification of CRISPR-Cas systems and a reconstruction of their evolution. In addition, we have extensively characterized protein domains and predicted numerous novel protein complexes involved in other forms of antivirus defense in prokaryotes. The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes, and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous over-represented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show non-random clustering in defense islands. It remains unclear to what extant these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic 'sinks' that accumulate diverse non-essential genes, particularly those acquired via HGT. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are non-randomly associated in islands, suggesting non-adaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.