PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain

[PSI(+)], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI(+)] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI(+)] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI(+)] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI(+)]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI(+)] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.     

Tentative identification of a resilin gene in Drosophila melanogaster

A search of the Drosophila genome for gene products with similarities to the amino acid sequences of three tryptic peptides from locust (Schistocerca gregaria) resilin gave two positive results: gene products CG15920 and CG9036. In both conceptual translation products a 62-residue region is present, which is identical to the resilin peptides in 29 positions. Gene product CG15920 has an amino acid composition closely resembling that of resilins from various insect species, and it has an N-terminal signal peptide sequence indicating that it is an extracellular protein. The 62-residue region shows similarity to the RR-2 sequence, which is common for a number of matrix proteins from insect solid cuticle. The N- and C-terminal regions flanking the 62-residue in CG15920 are dominated by 18 repeats of a 15-residue sequence and 11 repeats of a 13-residue sequence, respectively. The structures of the repeats predict that the peptide chain will fold in an irregular, extended beta-spiral, resembling the structures suggested for mammalian elastin and spider flagelliform silk, two materials which, like resilin, possess long-range elasticity. Accordingly, we suggest that gene product CG15920 is a Drosophila resilin precursor.     

A new repeat-masking method enables specific detection of homologous sequences

Biological sequences are often analyzed by detecting homologous regions between them. Homology search is confounded by simple repeats, which give rise to strong similarities that are not homologies. Standard repeat-masking methods fail to eliminate this problem, and they are especially ill-suited to AT-rich DNA such as malaria and slime-mould genomes. We present a new repeat-masking method, tantan, which is motivated by the mechanisms that create simple repeats. This method thoroughly eliminates spurious homology predictions for DNA–DNA, protein–protein and DNA–protein comparisons. Moreover, it enables accurate homology search for non-coding DNA with extreme A + T composition.     

Cluster of fibronectin type III repeats found in the human major histocompatibility complex class III region shows the highest homology with the repeats in an extracellular matrix protein, tenascin.

Walking and sequencing a genome portion centromeric of CYP21B in the human MHC class III region disclosed a cluster of fibronectin type III repeats in an approximately 50-kb DNA segment. Fibronectin type III repeats are known to consist of ca. 90 amino acid residues and exist in a wide range of protein species. Homology searches in protein databases showed that the repeats found had the highest homology with the repeats of human tenascin, an extracellular matrix protein. One cDNA sequence located immediately centromeric of CYP21B, the 3' portion of which is transcribed by the opposite strand of CYP21B, was found also to have six type III repeats followed by a fibrinogen domain. Pairwise homology comparison of these repeats in the MHC locus with those of human tenascin showed a general parallelism in their gene organization, indicating that the newly found repeats are elements of certain tenascin-like genea.     

Complete nucleotide sequence of mini-Rts1 and its copy mutant.

The nucleotide sequence of the whole mini-Rts1 genome consisting of 1,855 base pairs was determined. In addition to the cluster of five 24-base-pair direct repeats previously detected (Y. Kamio and Y. Terawaki, J. Bacteriol. 155:1185-1191, 1983), another cluster of three 21-base-pair direct repeats was found. All eight repeats were located in one direction and contained a consensus sequence TTCCCCPyPyPuPuCACACACC. Between the two clusters, a large open reading frame that could encode a 32,980-dalton polypeptide consisting of 288 amino acids was assigned. The molecular size predicted from the amino acid composition was close to the value of a unique mini-Rts1 product, the RepA protein, newly determined in minicells. A copy mutant of mini-Rts1 obtained in vitro by hydroxylamine treatment contained two-base-pair substitutions, one of which was located in the RepA protein coding region, and the other was close to the region where the oriC homologous sequences exist.     

The mechanism of RNA binding to TRAP: initiation and cooperative interactions

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 2'-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.     

Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins

Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein domains. Here we show that the FG domain of Nups from human, fly, worm, and other yeast species is also enriched in these disorder-associated AAs, indicating that structural disorder is a conserved feature of FG Nups and likely serves an important role in NPC function. Despite the conservation of AA composition, FG Nup sequences from different species show extensive divergence. A comparison of the AA substitution rates of proteins with syntenic orthologs in four Saccharomyces species revealed that FG Nups have evolved at twice the rate of average yeast proteins with most substitutions occurring in sequences between FG repeats. The rapid evolution of FG Nups is poorly explained by parameters known to influence AA substitution rate, such as protein expression level, interactivity, and essentiality; instead their rapid evolution may reflect an intrinsic permissiveness of natively unfolded structures to AA substitutions. The overall lack of AA sequence conservation in FG Nups is sharply contrasted by discrete stretches of conserved sequences. These conserved sequences highlight known karyopherin and nucleoporin binding sites as well as other uncharacterized sites that may have important structural and functional properties.     

ProtRepeatsDB: a database of amino acid repeats in genomes

Background  

Genome wide and cross species comparisons of amino acid repeats is an intriguing problem in biology mainly due to the highly polymorphic nature and diverse functions of amino acid repeats. Innate protein repeats constitute vital functional and structural regions in proteins. Repeats are of great consequence in evolution of proteins, as evident from analysis of repeats in different organisms. In the post genomic era, availability of protein sequences encoded in different genomes provides a unique opportunity to perform large scale comparative studies of amino acid repeats. ProtRepeatsDB is a relational database of perfect and mismatch repeats, access to which is designed as a resource and collection of tools for detection and cross species comparisons of different types of amino acid repeats.     

A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins.

We have identified a 102 kDa protein, p102, which is found on the cytoplasmic face of Golgi membranes, exocytic transport vesicles and in the cytosol. A monoclonal antibody that cross-reacts with p102 is able to immunoprecipitate a 500-600 kDa protein complex containing p102 and additional subunits. The composition of this p102-containing protein complex resembles that of the Golgi coatomer complex, which constitutes the coat of non-clathrin coated vesicles. One of the subunits of the p102 complex reacts with a monoclonal antibody that detects beta-COP, a subunit of the Golgi coatomer complex. Like beta-COP, p102 exists in a brefeldin A-sensitive association with Golgi membranes. The sequence of p102 contains an N-terminal domain composed of six repeats which are similar to those found in the beta subunit of trimeric G proteins and other regulatory proteins. We suggest that p102 may be involved in regulating membrane traffic in the constitutive exocytic pathway.     

Tandemly repeating peptide motifs and their secondary structure in Ceratitis capitata eggshell proteins Ccs36 and Ccs38.

Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide-X-X-X-Z-Z-Z-Z-Z- (where X = large hydrophobic residue and Z = beta-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel beta-pleated sheet structure of beta-sheet strands regularly alternating with beta-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different. The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide-Y-X-A-A-P-A-A-S- (X = G or S), whereas the N-terminal 'arm' contains repeats of the octapeptide -Z-Z-Z-A-X-A-A-Z- (X = Q, N or E and Z a beta-turn former). In both 'arms' alpha-helices are predicted, alternating with beta-turns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

Gram‐positive surface proteins can be covalently or non‐covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non‐identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock‐down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram‐positive bacteria.     

Domain structure and conserved epitopes of Sfb protein, the fibronectin-binding adhesin of Streptococcus pyogenes

Streptococcus pyogenes expresses a fibronectin-binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N-terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline-rich repeats which are flanked by non-repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin-binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N-terminal aromatic domain revealed similarities with carbohydrate-binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline-rich repeats of functionally distinct surface proteins from pathogenic Gram-positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin-binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N-termini of mature Sfb proteins, whereas sequences coding for the central and C-terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.     

A role for selection in regulating the evolutionary emergence of disease-causing and other coding CAG repeats in humans and mice

The evolutionary expansion of CAG repeats in human triplet expansion disease genes is intriguing because of their deleterious phenotype. In the past, this expansion has been suggested to reflect a broad genomewide expansion of repeats, which would imply that mutational and evolutionary processes acting on repeats differ between species. Here, we tested this hypothesis by analyzing repeat- and flanking-sequence evolution in 28 repeat-containing genes that had been sequenced in humans and mice and by considering overall lengths and distributions of CAG repeats in the two species. We found no evidence that these repeats were longer in humans than in mice. We also found no evidence for preferential accumulation of CAG repeats in the human genome relative to mice from an analysis of the lengths of repeats identified in sequence databases. We then investigated whether sequence properties, such as base and amino acid composition and base substitution rates, showed any relationship to repeat evolution. We found that repeat-containing genes were enriched in certain amino acids, presumably as the result of selection, but that this did not reflect underlying biases in base composition. We also found that regions near repeats showed higher nonsynonymous substitution rates than the remainder of the gene and lower nonsynonymous rates in genes that contained a repeat in both the human and the mouse. Higher rates of nonsynonymous mutation in the neighborhood of repeats presumably reflect weaker purifying selection acting in these regions of the proteins, while the very low rate of nonsynonymous mutation in proteins containing a CAG repeat in both species presumably reflects a high level of purifying selection. Based on these observations, we propose that the mutational processes giving rise to polyglutamine repeats in human and murine proteins do not differ. Instead, we propose that the evolution of polyglutamine repeats in proteins results from an interplay between mutational processes and selection.     

Evolution of subterminal satellite (StSat) repeats in hominids

Subterminal satellite (StSat) repeats, consisting of 32-bp-long AT-rich units (GATATTTCCATGTT(T/C)ATACAGATAGCGGTGTA), were first found in chimpanzee and gorilla (African great apes) as one of the major components of heterochromatic regions located proximal to telomeres of chromosomes. StSat repeats have not been found in orangutan (Asian great ape) or human. This patchy distribution among species suggested that the StSat repeats were present in the common ancestor of African great apes and subsequently lost in the lineage leading to human. An alternative explanation is that the StSat repeats in chimpanzee and gorilla have different origins and the repeats did not occur in human. The purpose of the present study was quantitative evaluation of the above alternative possibilities by analyzing the nucleotide variation contained in the repeats. We collected large numbers of sequences of repeat units from genome sequence databases of chimpanzee and gorilla, and also bonobo (an African great ape phylogenetically closer to chimpanzee). We then compared the base composition of the repeat units among the 3 species, and found statistically significant similarities in the base composition. These results support the view that the StSat repeats had already formed multiple arrays in the common ancestor of African great apes. It is thus suggested that humans lost StSat repeats which had once grown to multiple arrays.     

Evolutionary analysis of amino acid repeats across the genomes of 12 Drosophila species

Repeated motifs of amino acids within proteins are an abundant feature of eukaryotic sequences and may catalyze the rapid production of genetic and even phenotypic variation among organisms. The completion of the genome sequencing projects of 12 distinct Drosophila species provides a unique dataset to study these intriguing sequence features on a phylogeny with a variety of timescales. We show that there is a higher percentage of proteins containing repeats within the Drosophila genus than most other eukaryotes, including non-Drosphila insects, which makes this collection of species particularly useful for the study of protein repeats. We also find that proteins containing repeats are overrepresented in functional categories involving developmental processes, signaling, and gene regulation. Using the set of 1-to-1 ortholog alignments for the 12 Drosophila species, we test the ability of repeats to act as reliable phylogenetic signals and find that they resolve the generally accepted phylogeny despite the noise caused by their accelerated rate of evolution. We also determine that in general the position of repeats within a protein sequence is non-random, with repeats more often being absent from the middle regions of sequences. Finally we find evidence to suggest that the presence of repeats is associated with an increase in evolutionary rate upon the entire sequence in which they are embedded. With additional evidence to suggest a corresponding elevation in positive selection we propose that some repeats may be inducing compensatory substitutions in their surrounding sequence.     

Expression of selected domains of the circumsporozoite antigen ofPlasmodium knowlesi

The circumsporozoite antigen of the simian malarial parasite,Plqsmodium knowlesi, consists of tandemly repeated immunodominant peptide units which may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the whole of the non-repetitive region of this antigen has been expressed inEscherichia coli. The entire amino-terminal region up to the start of the repeats, and the full non-repetitive carboxyl region starting from the end of the repeats up to the termination codon, have been expressed separately, as fusion proteins with a 26 kD glutathione-S-transferase protein ofSchistosomq japonicum. A repeat-less truncated antigen has also been expressed as the same fusion protein. The amino-terminal fusion protein (GST-CSN), is a soluble protein of a molecular weight of 38 kD, which could be purified by affinity chromatography on immobilized glutathione. The carboxylterminal fusion protein (GST-CSC), is insoluble, migrates with an anomalous molecular weight of 32 kD, and binds to the affinity matrix weakly. The truncated repeat-less fusion protein (GST-CSNC) is also, an insoluble protein of molecular weight of 48 kD. Unlike the two separate domains, GST-CSNC is an extremely unstable protein inEscherichia coli.     

The US11 gene product of herpes simplex virus has intercellular trafficking activity.

The US11 gene product of herpes simplex virus is an abundant virion structural protein with RNA-binding regulatory activity. Its carboxyl-terminal half consists of tandem tripeptide repeats of the sequence RXP. We demonstrate that the US11 protein has intercellular trafficking activity and accumulates in the nucleolus when singly expressed in cultured cells, and that the RXP repeats are responsible for this activity. These same properties were also observed in cells expressing a fusion protein linking US11 to the green fluorescent protein. Furthermore, exogenous US11 protein was internalized by cells at 4 degrees C, which suggests that US11 protein uptake occurs primarily through an energy-independent pathway.