On How Many Fundamental Kinds of Cells are Present on Earth: Looking for Phylogenetic Traits that Would Allow the Identification of the Primary Lines of Descent

The phylogenetic analyses as far as the identification of the number of domains of life is concerned have not reached a clear conclusion. In the attempt to improve this circumstance, I introduce the concept that the amino acids codified in the genetic code might be of markers with outstanding phylogenetic power. In particular, I hypothesise the existence of a biosphere populated, for instance, by three groups of organisms having different genetic codes because codifying at least a different amino acid. Evidently, these amino acids would mark the proteins that are present in the three groups of organisms in an unambiguous way. Therefore, in essence, this mark would not be other than the one that we usually try to make in the phylogenetic analyses in which we transform the protein sequences in phylogenetic trees, for the purpose to identify, for example, the domains of life. Indeed, this mark would allow to classify proteins without performing phylogenetic analyses because proteins belonging to a group of organisms would be recognisable as marked in a natural way by at least a different amino acid among the diverse groups of organisms. This conceptualisation answers the question of how many fundamental kinds of cells have evolved from the Last Universal Common Ancestor (LUCA), as the genetic code has unique proprieties that make the codified amino acids excellent phylogenetic markers. The presence of the formyl-methionine only in proteins of bacteria would mark them and would identify these as domain of life. On the other hand, the presence of pyrrolysine in the genetic code of the euryarchaeota would identify them such as another fundamental kind of cell evolved from the LUCA. Overall, the phylogenetic distribution of formyl-methionine and pyrrolysine would identify at least two domains of life—Bacteria and Archaea—but their number might be actually four; that is to say, Bacteria, Euryarchaeota, archeobacteria that are not euryarchaeota and Eukarya. The usually accepted domains of life represented by Bacteria, Archaea and Eukarya are not compatible with the phylogenetic distribution of these two amino acids and therefore this last classification might be mistaken.     

Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus: a new type 2 RNase H with TBP-like substrate-binding domain at the N terminus

Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus stearothermophilus is a type 2 RNase H but shows poor amino acid sequence identity with another type 2 RNase H, RNase HII. It is composed of 310 amino acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal extension with unknown function and a unique active-site motif (DEDE), both of which are characteristics common to RNases HIII. To understand the role of these N-terminal extension and active-site residues, the crystal structure of Bst-RNase HIII was determined in both metal-free and metal-bound forms at 2.1-2.6 angstroms resolutions. According to these structures, Bst-RNase HIII consists of the N-terminal domain and C-terminal RNase H domain. The structures of the N and C-terminal domains were similar to those of TATA-box binding proteins and archaeal RNases HII, respectively. The steric configurations of the four conserved active-site residues were very similar to those of other type 1 and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98, and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli RNase HI, respectively. The mutational studies indicated that the replacement of either one of these residues with Ala resulted in a great reduction of the enzymatic activity. Overproduction, purification, and characterization of the Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter.     

Global phylogeny determined by the combination of protein domains in proteomes

The majority of proteins consist of multiple domains that are either repeated or combined in defined order. In this study, we survey the combination of protein domains defined at fold and fold superfamily levels in 185 genomes belonging to organisms that have been fully sequenced and introduce a method that reconstructs rooted phylogenomic trees from the content and arrangement of domains in proteins at a genomic level. We find that the majority of domain combinations were unique to Archaea, Bacteria, or Eukarya, suggesting most combinations originated after life had diversified. Domain repeat and domain repeat within multidomain proteins increased notably in eukaryotes, mainly at the expense of single-domain and domain-pair proteins. This increase was mostly confined to Metazoa. We also find an unbalanced sharing of domain combinations which suggests that Eukarya is more closely related to Bacteria than to Archaea, an observation that challenges the widely assumed eukaryote-archaebacterial sisterhood relationship. The occurrence and abundance of the molecular repertoire (interactome) of domain combinations was used to generate phylogenomic trees. These global interactome-based phylogenies described organismal histories satisfactorily, revealing the tripartite nature of life, and supporting controversial evolutionary patterns, such as the Coelomata hypothesis, the grouping of plants and animals, and the Gram-positive origin of bacteria. Results suggest strongly that the process of domain combination is not random but curved by evolution, rejecting the null hypothesis of domain modules combining in the absence of natural selection or an optimality criterion.     

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA

We recently provided the first report that RNase HIII can cleave a DNA-rN(1)-DNA/DNA substrate (rN(1), one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA-DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA-DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN(1)-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA-DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.     

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Ribonuclease (RNase) P is a site‐specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix‐loop‐helix protein‐binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 Å. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.     

A search method for homologs of small proteins. Ubiquitin-like proteins in prokaryotic cells?

The question of protein homology versus analogy arises when proteins share a common function or a common structural fold without any statistically significant amino acid sequence similarity. Even though two or more proteins do not have similar sequences but share a common fold and the same or closely related function, they are assumed to be homologs, descendant from a common ancestor. The problem of homolog identification is compounded in the case of proteins of 100 or less amino acids. This is due to a limited number of basic single domain folds and to a likelihood of identifying by chance sequence similarity. The latter arises from two conditions: first, any search of the currently very large protein database is likely to identify short regions of chance match; secondly, a direct sequence comparison among a small set of short proteins sharing a similar fold can detect many similar patterns of hydrophobicity even if proteins do not descend from a common ancestor. In an effort to identify distant homologs of the many ubiquitin proteins, we have developed a combined structure and sequence similarity approach that attempts to overcome the above limitations of homolog identification. This approach results in the identification of 90 probable ubiquitin-related proteins, including examples from the two prokaryotic domains of life, Archaea and Bacteria.