Functional unit of the RNA polymerase II C-terminal domain lies within heptapeptide pairs

Unlike all other RNA polymerases, the largest subunit (RPB1) of eukaryotic DNA-dependent RNA polymerase II (RNAP II) has a C-terminal domain (CTD) comprising tandemly repeated heptapeptides with the consensus sequence Y-S-P-T-S-P-S. The tandem structure, heptad consensus, and most key functions of the CTD are conserved between yeast and mammals. In fact, all metazoans, fungi, and green plants examined to date, as well as the nearest protistan relatives of these multicellular groups, contain a tandemly repeated CTD. In contrast, the RNAP II largest subunits from many other eukaryotic organisms have a highly degenerate C terminus or show no semblance of the CTD whatsoever. The reasons for intense stabilizing selection on CTD structure in certain eukaryotes, and its apparent absence in others, are unknown. Here we demonstrate, through in vivo genetic complementation, that the essential functional unit of the yeast CTD is contained within pairs of heptapeptides. Insertion of a single alanine residue between diheptads has little phenotypic effect, while increasing the distance between diheptads produces a mostly quantitative effect on yeast cell growth. We further explore structural constraints on the CTD within an evolutionary context and propose selective mechanisms that could maintain a global tandem structure across hundreds of millions of years of eukaryotic evolution.     

Evolution of the eukaryotic translation termination system: origins of release factors

Accurate translation termination is essential for cell viability. In eukaryotes, this process is strictly maintained by two proteins, eukaryotic release factor 1 (eRF1), which recognizes all stop codons and hydrolyzes peptidyl-tRNA, and eukaryotic release factor 3 (eRF3), which is an elongation factor 1alpha (EF-1alpha) homolog stimulating eRF1 activity. To retrace the evolution of this core system, we cloned and sequenced the eRF3 genes from Trichomonas vaginalis (Parabasalia) and Giardia lamblia (Diplomonada), which are generally thought to be "early-diverging eukaryotes," as well as those from two ciliates (Oxytricha trifallax and Euplotes aediculatus). We also determined the sequence of the eRF1 gene for G. lamblia. Surprisingly, the G. lamblia eRF3 appears to have only one domain, corresponding to EF-1alpha, while other eRF3s (including the T. vaginalis protein) have an additional N-terminal domain, of 66-411 amino acids. Considering this novel eRF3 structure and our extensive phylogenetic analyses, we suggest that (1) the current translation termination system in eukaryotes evolved from the archaea-like version, (2) eRF3 was introduced into the system prior to the divergence of extant eukaryotes, including G. lamblia, and (3) G. lamblia might be the first eukaryotic branch among the organisms considered.     

Phylogenetic place of mitochondrion-lacking protozoan, Giardia lamblia, inferred from amino acid sequences of elongation factor 2

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a mitochondrion-lacking protozoan, Giardia lamblia, were amplified by polymerase chain reaction, and their primary structures were analyzed. The deduced amino acid sequence was aligned with other eukaryotic and archaebacterial EF-2's, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) and the maximum parsimony (MP) methods. The ML analyses using six different models of amino acid substitutions and the MP analysis consistently suggest that among eukaryotic species being analyzed, G. lamblia is likely to have diverged from other higher eukaryotes on the early phase of eukaryotic evolution.      

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Protein phylogeny gives a robust estimation for early divergences of eukaryotes: phylogenetic place of a mitochondria-lacking protozoan, Giardia lamblia

A partial nucleotide sequence of the mRNA encoding a major part of elongation factor 1 alpha (EF1 alpha) from a mitochondria-lacking protozoan, Giardia lamblia, was reported, and the phylogenetic relationship among lower eukaryotes was inferred by the maximum- likelihood and maximum-parsimony methods of protein phylogeny. Both the methods consistently demonstrated that, G. lamblia among the four protozoan species being analyzed, is the earliest offshoot of the eukaryotic tree. Although the Giardia EF1 alpha gene showed an extremely high G+C content as compared with those of other protozoa, it was concentrated only at the third codon positions, resulting in no remarkable differences of amino acid frequencies vis-a-vis those of other species. This clearly suggests (a) that the amino acid frequencies of conservative proteins are free from the drastic bias of genome G+C content, which is a serious problem in the widely used tree of ribosomal RNA, and (b) that protein phylogeny gives a robust estimation for the early divergences in the evolution of eukaryotes.      

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.     

Core histones of the amitochondriate protist, Giardia lamblia

Genes coding for the core histones H2a, H2b, H3, and H4 of Giardia lamblia were sequenced. A conserved organism- and gene-specific element, GRGCGCAGATTTVGG, was found upstream of the coding region in all core histone genes. The derived amino acid sequences of all four histones were similar to their homologs in other eukaryotes, although they were among the most divergent members of this protein family. Comparative protein structure modeling combined with energy evaluation of the resulting models indicated that the G. lamblia core histones individually and together can assume the same three-dimensional structures that were established by X-ray crystallography for Xenopus laevis histones and the nucleosome core particle. Since G. lamblia represents one of the earliest-diverging eukaryotes in many different molecular trees, the structure of its histones is potentially of relevance to understanding histone evolution. The G. lamblia proteins do not represent an intermediate stage between archaeal and eukaryotic histones.     

RNA聚合酶监视的DNA修复机制

生物体在正常生命过程中面临内/外因来源的DNA损伤,DNA损伤不仅影响基因正确复制,也阻碍其正常转录. 为避免DNA损伤带来的灾难性后果,生物体进化出一整套修复机制,以保证复制和转录的正确性、基因组的完整性和遗传的稳定性. 本文重点综述了RNA聚合酶监视（RNA polymerase-surveilled,RNAP-S）的DNA修复机制. 首先从RNA聚合酶（RNA polymerase,RNAP）的结构出发介绍了RNAP对DNA损伤的感知机制;其次讨论了滞留RNAP的回溯、与其模板DNA的解离以及后续修复机制的启动,真核细胞科凯恩综合征B蛋白（Cockayne syndrome protein B,CSB）及其泛素化和8-氧代鸟嘌呤DNA糖基化酶1（8-oxoguanine DNA glycosylase1,OGG1）介导的RNAP-S修复;最后探讨了RNAP-S损伤修复的生物学意义并展望其前景.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.