Prediction of RNA secondary structure based on helical regions distribution

MOTIVATION: RNAs play an important role in many biological processes and knowing their structure is important in understanding their function. Due to difficulties in the experimental determination of RNA secondary structure, the methods of theoretical prediction for known sequences are often used. Although many different algorithms for such predictions have been developed, this problem has not yet been solved. It is thus necessary to develop new methods for predicting RNA secondary structure. The most-used at present is Zuker's algorithm which can be used to determine the minimum free energy secondary structure. However many RNA secondary structures verified by experiments are not consistent with the minimum free energy secondary structures. In order to solve this problem, a method used to search a group of secondary structures whose free energy is close to the global minimum free energy was developed by Zuker in 1989. When considering a group of secondary structures, if there is no experimental data, we cannot tell which one is better than the others. This case also occurs in combinatorial and heuristic methods. These two kinds of methods have several weaknesses. Here we show how the central limit theorem can be used to solve these problems. RESULTS: An algorithm for predicting RNA secondary structure based on helical regions distribution is presented, which can be used to find the most probable secondary structure for a given RNA sequence. It consists of three steps. First, list all possible helical regions. Second, according to central limit theorem, estimate the occurrence probability of every helical region based on the Monte Carlo simulation. Third, add the helical region with the biggest probability to the current structure and eliminate the helical regions incompatible with the current structure. The above processes can be repeated until no more helical regions can be added. Take the current structure as the final RNA secondary structure. In order to demonstrate the confidence of the program, a test on three RNA sequences: tRNAPhe, Pre-tRNATyr, and Tetrahymena ribosomal RNA intervening sequence, is performed. AVAILABILITY: The program is written in Turbo Pascal 7.0. The source code is available upon request. CONTACT: Wujj@nic.bmi.ac.cn or Liwj@mail.bmi.ac.cn     

Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.     

Phylogenetic analysis of tmRNA secondary structure

The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.     

The graduation of secondary structure elements

The yield of bone scintigraphy was assessed in a general hospital. A random 1:2 sample of the bone studies performed within a year was evaluated. The referral forms were judged to show absent, ambiguous or clear diagnostic questions. The indications for which the study was ordered were matched with the scintigraphic diagnosis, and yield was defined as the proportion of studies with relevant findings on scintigraphy. The overall yield was 23.5%. In cancer patients the yield was 27.2% and in repeat studies 40.2%. In non-cancer patients the yield was 19.8% and when scintigraphy was performed for the evaluation of specific disease entities it was 35.7%. Relatively low-yield indications in non-cancer patients were suspected metastases 11.1%, and evaluation of X-ray abnormalities 11.5%. An association between the clarity of the diagnostic question and the scintigraphic yield was detected in non-cancer patients. When a diagnostic question was absent the yield was 12%, ambiguous 19%, and when it was clear 26%. This correlation is probably attributed to better understanding of this common procedure, and better definition of the diagnostic question in patients with diverse clinical conditions as compared to cancer patients in whom the question always relates to the presence of bone metastases.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

Investigations on the primary structure of human plasminogen. Further evidence for sequence homology

A relatively rare benign tumor of the fallopian tube is presented, i.e. an intramural leiomyoma, which was incidentally found during histologic examination of the tubal isthmus excised for sterilization. The literature is briefly reviewed.     

Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.     

Protein secondary structure prediction by the analysis of variation and conservation in multiple alignments

A number of methods exist for the prediction of protein secondary structure from primary sequence. One method identifies variable charged and conserved hydrophobic residues within large multiple alignments as a means of indicating outside and inside sites respectively in the protein structure. These sites are then manually fitted to secondary structure templates to generate a secondary structure prediction. Using the existing theoretical bases of this method, we present an algorithm (STAMA) which automatically carries out the initial variation/conservation analysis of the alignment. We also test the accuracy of complete predictions carried out by manual fitting of the STAMA-derived assignments to structure templates, using five large multiple alignments each including a protein of known structure. The method was found on average to predict only 57% of residues in the correct secondary structure, and was only as accurate as predictions carried out using the established and automated method of Garnier, Osguthorpe and Robson (1978) applied to a single sequence. When used in conjunction with other secondary structure prediction methods, however, the resulting consensus predictions were found to be very accurate, with 78% of the elements (alpha helices or beta strands) for which a consensus could be obtained being predicted correctly. The algorithm presented here, plus the assessment of the accuracy of prediction generated by this method, should enable this predictive approach to receive informed general use.     

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.     

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases

BACKGROUND: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.     

Prediction of the crystal structure types of equiatomic ternary silicides and germanides

New unsynthesized equiatomic ABX (A and B are various metals; X = Si or Ge) compounds are predicted, and their types of crystal structure are forecasted under standard conditions. Only the data on the properties of the elements—components of compounds are used for their prediction. The calculations are performed using a special-purpose software package of computer analysis of information intended for searching for regularities in databases on the properties of inorganic compounds, and this package is based on the methods of precedent pattern recognition. Computer analysis of the data on the well-known compounds shows that the functions that are most important for the classification of systems in the sign of formation or absence of equiatomic compounds are M(A) × M(B) and I(A) × I(X), where M is the Mendeleev-Pettifor number of element A or B and I is the thermal conductivity of element A or X. The parameters that most strongly separate compounds for crystal chemical classification are functions T(A) + T(B) (where T is the melting temperature of element A or B), I(A), M(A) × M(B), and I(A) × I(X).     

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films

HP1 integrase promotes site-specific recombination of the HP1 genome into that of Haemophilus influenzae. The isolated C-terminal domain (residues 165-337) of the protein interacts with the recombination site and contains the four catalytic residues conserved in the integrase family. This domain represents a novel fold consisting principally of well-packed alpha helices, a surface beta sheet, and an ordered 17-residue C-terminal tail. The conserved triad of basic residues and the active-site tyrosine are contributed by a single monomer and occupy fixed positions in a defined active-site cleft. Dimers are formed by mutual interactions of the tail of one monomer with an adjacent monomer; this orients active-site clefts antiparallel to each other.     

Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. Nup475 contains two tandemly repeated sequences YKTELCX8CX5CX3H (Cys3His repeats) that are thought to be zinc-bindin domains. Similar sequences have been found in a number of proteins from various species of eukaryotes. To determine the metal binding properties and secondary structure of the putative zinc-binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences. The peptide with a single domain bound 1.0 +/- 0.1 equivalents of Co2+, and the peptide with two domains bound 1.7 +/- 0.4 equivalents of Co2+. Both peptides bound Co2+ and Zn2+ with affinities similar to those of classical zinc finger peptides. In each case, the Co2+ complex exhibited strong d-d transitions characteristic of tetrahedral coordination. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys3His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys3His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.