Consensus Secondary Structure Research Articles

Primary structure of two cytolysin isoforms from Stichodactyla helianthus differing in their hemolytic activity.

Sticholysin I (St-I) and sticholysin II (St-II) are cytolysins purified from the sea anemone Stichodactyla helianthus with a high degree of sequence identity (93%) but clearly differenced in their hemolytic activity. In order to go further into the structural determinants for the different behavior of St-I and St-II, we report here the complete amino acid sequences and the consensus secondary structure prediction of both proteins. The complete determination of St-II primary structure confirms the partial revision of cytolysin III amino acid sequence. All nonconservative changes between St-I and St-II are located at the N-terminal. According to our prediction these changes could be located at the same face of an α-helix during pore formation events and could account for the observed differences in hemolytic activity between St-I and St-II.

Secondary structural and phylogenetic implications of nuclear large subunit ribosomal RNA in the ectomycorrhizal fungus Tricholoma matsutake.

The sequence of large subunit (LSU) and 5.8S rRNA genes has been determined for Tricholoma matsutake. A secondary structure model was predicted for both LSU and 5.8S rRNAs, showing most of the structural features consistent with those of the consensus secondary structure model proposed for the eukaryotic cytoplasmic LSU rRNAs. With a reconstructed eukaryotic phylogeny based on full-length LSU rDNA sequences, T. matsutake was placed on the same branch with Cryptococcus neoformans as its closest neighbor. We proposed that T. matsutake be considered as one of the representative members of the division Basidiomycota. Here we report for the first time the complete LSU rRNA gene sequence in T. matsutake, a member of Homobasidiomycetes.

Decision tree-based formation of consensus protein secondary structure prediction.

Prediction of protein secondary structure provides information that is useful for other prediction methods like fold recognition and ab initio 3D prediction. A consensus prediction constructed from the output of several methods should yield more reliable results than each of the individual methods. We present an approach that reveals subtle but systematic differences in the output of different secondary structure prediction methods allowing the derivation of coherent consensus predictions. The method uses a machine learning technique that builds decision trees from existing data. The first results of our analysis show that consensus prediction of protein secondary structure may be improved both quantitatively and qualitatively.

Pyrimidine-Rich Region Mutations Compensate for a Stem-Loop V Lesion in the 5′ Noncoding Region of Poliovirus Genomic RNA

Five revertants of a linker-scanning mutation adjacent to the stem-loop V attenuation determinant (X472) in the 5′ noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs with seven nucleotide changes in the pyrimidine-rich region. Generation of the identical rare second-site mutations suggests the existence of a replicase-dependent mutagenesis mechanism during poliovirus replication. Enzymatic structure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary structure model is presented for wild-type stem-loops V and VI and the pyrimidine-rich region located in the 5′ noncoding region of poliovirus RNA. A pyrimidine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences were not due to cell-type differences in viral translation or RNA replication. Release of X472-R4N from HeLa cells was 10-fold lower than release from neuroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently from neuroblastoma cells (∼4-fold increase compared with release from HeLa cells), indicating that poliovirus neurotropism may be influenced by the cell-type efficiency of virus release. Thermal treatment increased the levels of infectious X472-R4N virions but not wild-type virus particles; thus RNA sequence and structural changes in the mutated 5′ noncoding region of X472-R4N may have altered RNA–protein interactions necessary for virus infectivity.

A new method to predict the consensus secondary structure of a set of unaligned RNA sequences.

To predict the consensus secondary structure, possibly including pseudoknots, of a set of RNA unaligned sequences. We have designed a method based on a new representation of any RNA secondary structure as a set of structural relationships between the helices of the structure. We refer to this representation as a structural pattern. In a first step, we use thermodynamic parameters to select, for each sequence, the best secondary structures according to energy minimization and we represent each of them using its corresponding structural pattern. In a second step, we search for the repeated structural patterns, i.e. the largest structural patterns that occur in at least one sequence, i.e. included in at least one of the structural patterns associated to each sequence. Thanks to an efficient encoding of structural patterns, this search comes down to identifying the largest repeated word suffixes in a dictionary. In a third step, we compute the plausibility of each repeated structural pattern by checking if it occurs more frequently in the studied sequences than in random RNA sequences. We then suppose that the consensus secondary structure corresponds to the repeated structural pattern that displays the highest plausibility. We present several experiments concerning tRNA, fragments of 16S rRNA and 10Sa RNA (including pseudoknots); in each of them, we found the putative consensus secondary structure.

Molecular characterization and genome organization of 7SL RNA genes from hop (Humulus lupulus L.).

A wide spectrum of hop 7SL RNA-encoding sequences was detected by temperature gradient-gel electrophoresis. Four hop 7SL RNA genes were cloned and characterized. A new subvariant of the upstream sequence element (USE) 5'TCCCACATGG 3' and two distinct variants of TATA signal were found at positions characteristic for RNA polymerase III-driven transcription in plants. In addition, a more distant conserved sequence element 5' CATGTATAAACTTTCTGC 3' was present in all cloned genes, about 160 bp upstream of the 7SL RNA coding sequence. Consensus secondary structures calculated for hop 7SL RNAs revealed characteristic features, although some structure differences from formerly published models were predicted. Specific in-vitro transcription of plant 7SL RNA genes was observed in a heterologous system (HeLa extract). This in-vitro transcription assay showed significant differences among individual clones in transcription rates, suggesting the requirement of complexity of 7SL RNA sequence for its efficient transcription in HeLa extract. Southern blot analysis of hop DNA revealed 12 7SL-specific signals corresponding to HindIII fragments ranging from 0.45 to 7.8 kb. Several 7SL RNA-encoding sequences and various intergenic spacers were amplified from the individual HindIII fragments of about 1.3 and 2.8 kb. These facts suggest that at least some of the hop 7SL RNA genes are organized in genomic clusters.

Improved Secondary Structure Predictions for a Nicotinic Receptor Subunit: Incorporation of Solvent Accessibility and Experimental Data into a Two-Dimensional Representation

Abstract A refined prediction of the nicotinic acetylcholine receptor (nAChR) subunits’ secondary structure was computed with third-generation algorithms. The four selected programs, PHD, Predator, DSC, and NNSSP, based on different prediction approaches, were applied to each sequence of an alignment of nAChR and 5-HT 3 receptor subunits, as well as a larger alignment with related subunit sequences from glycine and GABA receptors. A consensus prediction was computed for the nAChR subunits through a “winner takes all” method. By integrating the probabilities obtained with PHD, DSC, and NNSSP, this prediction was filtered in order to eliminate the singletons and to more precisely establish the structure limits (only 4% of the residues were modified). The final consensus secondary structure includes nine α-helices (24.2% of the residues, with an average length of 13.9 residues) and 17 β-strands (22.5% of the residues, with an average length of 6.6 residues). The large extracellular domain is predicted to be mainly composed of β-strands, with only two helices at the amino-terminal end. The transmembrane segments are predicted to be in a mixed α/ β topology (with a predominance of α-helices), with no known equivalent in the current protein database. The cytoplasmic domain is predicted to consist of two well-conserved amphipathic helices joined together by an unfolded stretch of variable length and sequence. In general, the segments predicted to occur in a periodic structure correspond to the more conserved regions, as defined by an analysis of sequence conservation per position performed on 152 superfamily members. The solvent accessibility of each residue was predicted from the multiple alignments with PHDacc. Each segment with more than three exposed residues was assumed to be external to the core protein. Overall, these data constitute an envelope of structural constraints. In a subsequent step, experimental data relative to the extracellular portion of the complete receptor were incorporated into the model. This led to a proposed two-dimensional representation of the secondary structure in which the peptide chain of the extracellular domain winds alternatively between the two interfaces of the subunit. Although this representation is not a tertiary structure and does not lead to predictions of specific β- β interaction, it should provide a basic framework for further mutagenesis investigations and for fold recognition (threading) searches.

Fold recognition using sequence and secondary structure information

We applied a succession of sequence search and structure prediction methods to the targets in the fold recognition part of the CASP3 experiment. For each target, we expanded an initial sequence space, obtained through PSI-BLAST, by searching for statistically significant relationships to low-scoring sequences and then by searching for conserved sequence patterns. We then divided the proteins in the sequence space into families and built an alignment hierarchically, using the multiple alignment program MACAW. If no significant similarity to a protein of known structure was apparent at this point, we submitted the alignment to the Jpred server for consensus secondary structure prediction and searched the structure space using the secondary structure mapping program MAP. Failing this, we compared the structural properties that we believed we recognized in the aligned proteins to the folds in the SCOP database, using visual inspection. If all these methods failed to uncover a plausible match, we predicted that the target would adopt a novel fold. This procedure yielded correct answers for seven of twenty-one targets and a partly correct answer for one. A retrospective analysis shows that automating the sequence search procedures would have represented a significant improvement, with at least three additional correct predictions.

Fold recognition using sequence and secondary structure information

We applied a succession of sequence search and structure prediction methods to the targets in the fold recognition part of the CASP3 experiment. For each target, we expanded an initial sequence space, obtained through PSI-BLAST, by searching for statistically significant relationships to low-scoring sequences and then by searching for conserved sequence patterns. We then divided the proteins in the sequence space into families and built an alignment hierarchically, using the multiple alignment program MACAW. If no significant similarity to a protein of known structure was apparent at this point, we submitted the alignment to the Jpred server for consensus secondary structure prediction and searched the structure space using the secondary structure mapping program MAP. Failing this, we compared the structural properties that we believed we recognized in the aligned proteins to the folds in the SCOP database, using visual inspection. If all these methods failed to uncover a plausible match, we predicted that the target would adopt a novel fold. This procedure yielded correct answers for seven of twenty-one targets and a partly correct answer for one. A retrospective analysis shows that automating the sequence search procedures would have represented a significant improvement, with at least three additional correct predictions.

RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability.

In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.

Turnip yellow mosaic virus isolates with experimentally produced recombinant virion proteins.

The consensus secondary structure of the virion proteins (VPs) of tymoviruses was predicted from their amino acid sequences using a combination of computer methods: profile alignment, hydrophobicity moment and 'PredictProtein'. All methods predicted that they were eight-stranded anti-parallel beta-barrels with two alpha-helical regions. The predicted structure was used to design recombinants of turnip yellow mosaic virus (TYMV) in which selected parts of its VP were replaced with homologous regions of belladonna mottle virus (BeMV) in a cDNA clone encoding the genome of TYMV. Six of ten such recombinants were fully viable and most gave symptoms in Chinese cabbage indistinguishable from those of TYMV, although they did not always infect plants systemically and none infected hosts of BeMV or of other tymoviruses. A TYMV recombinant with the N-terminal part of its VP replaced with the E71 epitope of Plasmodium falciparum was also viable, but others with the same region replaced with the V3 region of the lentivirus human immunodeficiency virus type 1 were not. Epitope analysis of antisera prepared against the virions of parental TYMV and some of the recombinants showed that, although the N terminus of the VP is immunogenically dominant, it is not exposed at the surface of the virion, a finding confirmed by comparing the electrophoretic mobilities of the virions. The recently published structure of the TYMV VP determined by X-ray crystallography confirms the accuracy of the predicted secondary structure of the VP, and hence the utility of the methods used.

JPred: a consensus secondary structure prediction server.

An interactive protein secondary structure prediction Internet server is presented. The server allows a single sequence or multiple alignment to be submitted, and returns predictions from six secondary structure prediction algorithms that exploit evolutionary information from multiple sequences. A consensus prediction is also returned which improves the average Q3 accuracy of prediction by 1% to 72.9%. The server simplifies the use of current prediction algorithms and allows conservation patterns important to structure and function to be identified. http://barton.ebi.ac.uk/servers/jpred.h tml geoff@ebi.ac.uk

Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.

The RNA Subunit of Ribonuclease P from the Zebrafish, Danio rerio

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

Molecular Evolution of the C-terminal Cytoplasmic Domain of a Superfamily of Bacterial Receptors Involved in Taxis

Twenty-nine proteins from 16 different species of prokaryotes revealed an extensive sequence homology with the cytoplasmic domain of the Escherichia coliaspartate receptor. The high percentage of identity indicated that they constitute a superfamily of proteins. A consensus secondary structure consisting mostly of α-helices was predicted. The occurrence of a seven-residue repeat ( a-b-c-d-e-f-g), in which both the aand dresidues were hydrophobic with few exceptions, provided additional evidence for a conserved α-helical conformation. Sequence alignments, together with the predicted secondary structure, led to identification of the boundaries for the functional units constituting the cytoplasmic domain. Putative methylation sites were assigned for all the members of this superfamily. These proteins could be grouped into three classes based on the presence of 14-residue insertion/deletion regions found within both the signalling and the methylation functional units of the cytoplasmic domain. The gene coding for the C-terminal cytoplasmic domain of these proteins apparently evolved through gene duplication from a common ancestor in which the four original 14-residue insertion/deletion regions were deleted two by two during evolution.

The protein fold of the hyaluronate-binding proteoglycan tandem repeat domain of link protein, aggrecan and CD44 is similar to that of the C-type lectin superfamily

Link protein and aggrecan of the extracellular matrix each contain two proteoglycan tandem repeat (PTR) domains that interact with hyaluronate. Consensus secondary structure predictions for 59 PTR sequences and 129 C-type lectin sequences give similar patterns of two a-helices and up to seven β-strands. Protein fold recognition analyses show that the 59 PTR sequences are highly compatible with the C-type lectin crystal structure. The predicted fold consists of a conserved motif formed from an antiparallel β-sheet flanked by two α-helices, the motif being attached to two distinct types of sheet region in the two superfamilies. Arg9 or Lys11 on an exposed loop and up to three other Arg residues in the β-sheet region are conserved and may form part of a hyaluronate binding site.

18] Identifying consensus patterns and secondary structure in SELEX sequence sets

The general methodology presented for the analysis of SELEX (Systematic Evolution of Ligands by EXponential enrichment) isolated molecules relies on the power of consensus to guide multiple sequence alignment and identify common secondary structure. The information derived from consensus features among functionally related sequences is vastly greater than that obtained from single sequences. Computational tools have been developed that take advantage of consensus-derived information for the purpose of identifying the structural motifs responsible for the observed activity. Relatively high stringency is imposed on deriving the initial consensus patterns and structures, from which common motifs can be defined. Once a primary and secondary structural motif is obtained, sequences more distantly related may be identified by taking both sequence and structure into account for alignment. Ultimately, these data will be used to produce consensus 3D structural models for a set of molecules, with common functional properties. Such structural information is important for understanding and modifying the interactions critical for activity.

Inhibitory DNA ligands to platelet-derived growth factor B-chain.

We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (Kd approximately 10(-10) M) and to PDGF-AA with lower affinity (> 10(-8) M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2'-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of 125I-PDGF-BB but not of 125I-PDGF-AA to PDGF alpha- or beta-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of approximately 1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [3H]thymidine incorporation in PAE cells expressing the PDGF beta-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

β-Sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy

Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three β-strands was predicted. Sequence threading against known protein folds indicated consistency with small β-sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT-IR spectroscopy on this indicated a small amount of β-sheet together with turns and loops. LDLr is proposed to have a β-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.