Generalized electrostatic model of the wrapping of DNA around oppositely charged proteins

Histonelike proteins in prokaryotes and histone octamers in eukaryotes carry large positive charges, which are responsible of strong electrostatic interactions with DNA. As a result, DNA wraps around proteins and genetic information is condensed. We describe a generalized model of these electrostatic interactions mediated by salt that explains the wrapping of DNA around the nucleosome octamer, around remodeling factors in eukaryotes and around histonelike proteins in prokaryotes. It comes out that small changes in protein dimension and charge produce large effects in the supramolecular DNA-protein architecture.     

Signs of Ancient and Modern Exon-Shuffling Are Correlated to the Distribution of Ancient and Modern Domains Along Proteins

Exon-shuffling is an important mechanism accounting for the origin of many new proteins in eukaryotes. However, its role in the creation of proteins in the ancestor of prokaryotes and eukaryotes is still debatable. Excess of symmetric exons is thought to represent evidence for exon-shuffling since the exchange of exons flanked by introns of the same phase does not disrupt the reading frame of the host gene. In this report, we found that there is a significant correlation between symmetric units of shuffling and the age of protein domains. Ancient domains, present in both prokaryotes and eukaryotes, are more frequently bounded by phase 0 introns and their distribution is biased towards the central part of proteins. Modern domains are more frequently bounded by phase 1 introns and are present predominantly at the ends of proteins. We propose a model in which shuffling of ancient domains mainly flanked by phase 0 introns was important in the ancestor of eukaryotes and prokaryotes, during the creation of the central part of proteins. Shuffling of modern domains, predominantly flanked by phase 1 introns, accounted for the origin of the extremities of proteins during eukaryotic evolution.     

Comparing function and structure between entire proteomes

More than 30 organisms have been sequenced entirely. Here, we applied a variety of simple bioinformatics tools to analyze 29 proteomes for representatives from all three kingdoms: eukaryotes, prokaryotes, and archaebacteria. We confirmed that eukaryotes have relatively more long proteins than prokaryotes and archaes, and that the overall amino acid composition is similar among the three. We predicted that approximately 15%-30% of all proteins contained transmembrane helices. We could not find a correlation between the content of membrane proteins and the complexity of the organism. In particular, we did not find significantly higher percentages of helical membrane proteins in eukaryotes than in prokaryotes or archae. However, we found more proteins with seven transmembrane helices in eukaryotes and more with six and 12 transmembrane helices in prokaryotes. We found twice as many coiled-coil proteins in eukaryotes (10%) as in prokaryotes and archaes (4%-5%), and we predicted approximately 15%-25% of all proteins to be secreted by most eukaryotes and prokaryotes. Every tenth protein had no known homolog in current databases, and 30%-40% of the proteins fell into structural families with >100 members. A classification by cellular function verified that eukaryotes have a higher proportion of proteins for communication with the environment. Finally, we found at least one homolog of experimentally known structure for approximately 20%-45% of all proteins; the regions with structural homology covered 20%-30% of all residues. These numbers may or may not suggest that there are 1200-2600 folds in the universe of protein structures. All predictions are available at http://cubic.bioc.columbia.edu/genomes.     

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.     

Functional importance of RNA interactions in selection of translation initiation codons

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis -acting mRNA elements and trans -acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.     

Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells

It is well established that hydrophobic signal sequences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell membranes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention. To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacterial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been examined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post-translationally modified before secretion. Data presented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenomenon and infer that there are no specific requirements with respect to the origin of the signal sequences.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Natural transformation increases the rate of adaptation in the human pathogen Helicobacter pylori

Gene exchange between individuals can lead to profound evolutionary effects at both the genomic and population levels. These effects have sparked widespread interest in examining the specific adaptive benefits of recombination. Although this work has primarily focused on the benefits of sex in eukaryotes, it is assumed that similar benefits of genetic exchange apply across eukaryotes and prokaryotes. Here we report a direct test of this assumption using the naturally transformable human gastric pathogen Helicobacter pylori as a model organism. We show that genetic exchange accelerates adaptation to a novel laboratory environment within bacterial populations and that a general adaptive advantage exists for naturally transformable strains when transfer occurs among conspecific backgrounds. This finding demonstrates that there are generalized benefits to adaptation in both eukaryotes and prokaryotes even though the underlying processes are mechanistically different.     

The Ancient Microbial RIO Kinases

The RIO kinases existed before the split between Archaea and Eubacteria and are essential in eukaryotes. Although much has been elucidated in the past few years regarding the function of these proteins in eukaryotes, questions remain about their role in prokaryotes. Comparison of structure and sequence suggests that the ancient RIO kinases may have similar functional properties in prokaryotes as they do in eukaryotes. The conservation of charge distribution, functional residues, and overall structure supports a role for these proteins in ribosome interactions, as is their purpose in eukaryotes. However, a lack of study in this area has left little direct evidence in support of this function.     

Structure and expression of the Zea mays mutS-homologs Mus1 and Mus2

DNA mismatch repair proteins play an important role in maintaining the integrity of the genetic information during replication and homologous recombination. The MutS-homologous (MSH) and MutL-homologous (MLH) proteins are highly conserved among all prokaryotes and eukaryotes. We have isolated two mutS homologous genes from Zea mays, named Mus1 and Mus2. Phylogenetic analysis identifies Mus1 as a member of the MSH2 protein family. Mus2 is an ortholog of the Arabidopsis thaliana MSH7 protein and belongs to a subgroup of MSH proteins that is possibly plant-specific. Mus1 and Mus2 are expressed at very low levels. Mus1 is located on chromosome 7L near locus b32B, and mus2 maps on chromosome 3S.     

Regulation of Hed1 and Rad54 binding during maturation of the meiosis‐specific presynaptic complex

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double‐strand break (DSB) repair, and the meiosis‐specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis‐specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real‐time single‐molecule imaging to probe how the Hed1‐ and Rad54‐mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.     

The Caenorhabditis elegansRAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings.     

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.     

Seeking the determinants of the elusive functions of Sco proteins

Sco proteins are present in all types of organisms, including the vast majority of eukaryotes and many prokaryotes. It is well established that Sco proteins in eukaryotes are involved in the assembly of the Cu(A) cofactor of mitochondrial cytochrome c oxidase; however their precise role in this process has not yet been elucidated at the molecular level. In particular, some but not all eukaryotes including humans possess two Sco proteins whose individual functions remain unclear. There is evidence that eukaryotic Sco proteins are also implicated in other cellular processes such as redox signalling and regulation of copper homeostasis. The range of physiological functions of Sco proteins appears to be even wider in prokaryotes, where Sco-encoding genes have been duplicated many times during evolution. While some prokaryotic Sco proteins are required for the biosynthesis of cytochrome c oxidase, others are most likely to take part in different processes such as copper delivery to other enzymes and protection against oxidative stress. The detailed understanding of the multiplicity of roles ascribed to Sco proteins requires the identification of the subtle determinants that modulate the two properties central to their known and potential functions, i.e. copper binding and redox properties. In this review, we provide a comprehensive summary of the current knowledge on Sco proteins gained by genetic, structural and functional studies on both eukaryotic and prokaryotic homologues, and propose some hints to unveil the elusive molecular mechanisms underlying their functions.     

New insights into the molecular mechanisms of evolution: stress increases genetic diversity

The mechanisms of stress-induced mutagenesis in prokaryotes and realization of reserved (preaccumulated) genetic variation in eukaryotes are considered. In prokaryotes, replication becomes error-prone in stress because of the induction of the SOS response and the inactivation of the mismatch repair system; stress also increases the transposition rate and the efficiency of interspecific gene transfer. In eukaryotes, chaperone HSP90, which restores the native folding of mutant proteins (e.g., signal transduction and morphogenetic proteins) in normal conditions, fails to do so in stress, which leads to abrupt expression of multiple mutations earlier reserved in the corresponding genes. The role of these mechanisms in the evolution of prokaryotes and eukaryotes is discussed.     

Right or left turn? RecA family protein filaments promote homologous recombination through clockwise axial rotation

The RecA family proteins mediate homologous recombination, a ubiquitous mechanism for repairing DNA double-strand breaks (DSBs) and stalled replication forks. Members of this family include bacterial RecA, archaeal RadA and Rad51, and eukaryotic Rad51 and Dmc1. These proteins bind to single-stranded DNA at a DSB site to form a presynaptic nucleoprotein filament, align this presynaptic filament with homologous sequences in another double-stranded DNA segment, promote DNA strand exchange and then dissociate. It was generally accepted that RecA family proteins function throughout their catalytic cycles as right-handed helical filaments with six protomers per helical turn. However, we recently reported that archaeal RadA proteins can also form an extended right-handed filament with three monomers per helical turn and a left-handed protein filament with four monomers per helical turn. Subsequent structural and functional analyses suggest that RecA family protein filaments, similar to the F1-ATPase rotary motor, perform ATP-dependent clockwise axial rotation during their catalytic cycles. This new hypothesis has opened a new avenue for understanding the molecular mechanism of RecA family proteins in homologous recombination.     

New Insights into the Molecular Mechanisms of Evolution: Stress Increases Genetic Diversity

The mechanisms of stress-induced mutagenesis in prokaryotes and realization of reserved (preaccumulated) genetic variation in eukaryotes are considered. In prokaryotes, replication becomes error-prone in stress because of the induction of the SOS response and the inactivation of the mismatch repair system; stress also increases the transposition rate and the efficiency of interspecific gene transfer. In eukaryotes, chaperone HSP90, which restores the native folding of mutant proteins (e.g., signal transduction and morphogenetic proteins) in normal conditions, fails to do so in stress, which leads to abrupt expression of multiple mutations earlier reserved in the corresponding genes. The role of these mechanisms in the evolution of prokaryotes and eukaryotes is discussed.     

Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family

Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes.     

Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes

The basic features of the active filaments that use nucleotide hydrolysis to organise the cytoplasm are remarkably similar in the majority of all cells and are either actin-like or tubulin-like. Nearly all prokaryotic cells contain at least one form of FtsZ, the prokaryotic homologue of tubulin and some bacterial plasmids use tubulin-like TubZ for segregation. The other main family of active filaments, assembled from actin-like proteins, occurs in a wide range of bacterial species as well as in all eukaryotes. Some bacterial plasmids also use ParM, another actin-like protein. Higher-order filament structures vary from simple to complex depending on the cellular application. Equally, filament-associated proteins vary greatly between species and it is not possible currently to trace their evolution from prokaryotes to eukaryotes. This lack of similarity except in the three-dimensional structures and longitudinal interactions between the filament subunits hints that the most basic cellular function of the filaments is to act as linear motors driven by assembly dynamics and/or bending and hence we term these filament systems 'cytomotive'. The principle of cytomotive filaments seems to have been invented independently for actin- and tubulin-like proteins. Prokaryotes appear to have a third class of cytomotive filaments, typically associated with surfaces such as membranes or DNA: Walker A cytoskeletal ATPases (WACA). A possible evolutionary relationship of WACAs with eukaryotic septins is discussed.