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Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.

Recent developments in the study of RNA silencing indicate that double-stranded RNA (dsRNA) can be used in eukaryotes to block expression of a corresponding cellular gene. There is also a large class of small non-coding RNAs having potential to form a distinct, stable stem-loop in numbers of eukaryotic genomes. We had reported that a large imperfect dsRNA structure with hundreds of base-pairs (bp) in the 3' untranslated region (3' UTR) of cytotoxic ribonuclease was correlated with the translation suppression. In this study, we search for such dsRNAs in a 3' UTR database. The occurrence rate of large dsRNA in 3' UTRs ranges from 0.01% in plant to 0.30% in vertebrate mRNAs. However, small imperfect dsRNAs of ~ 30 bp are much more prevalent than large ones. The small dsRNAs are statistically very significant and uniquely well-ordered. Most of them have the conserved structural features of pre-miRNAs. Our data mining of the dsRNAs in the 3' UTR database can be used to explore RNA-based regulation of gene expression.

Distinct RNA structures are frequently involved in a wide-range of functions in various biological mechanisms. The three dimensional RNA structures solved by X-ray crystallography and various well-established RNA phylogenetic structures indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop region. Discovery of well-ordered RNA structures and their homologues in genome-wide searches will enhance our ability to detect the RNA structural motifs and help us to highlight their association with functional and regulatory RNA elements. We present here a novel computer algorithm, HomoStRscan, that takes a single RNA sequence with its secondary structure to search for homologous-RNAs in complete genomes. This novel algorithm completely differs from other currently used search algorithms of homologous structures or structural motifs. For an arbitrary segment (or window) given in the target sequence, that has similar size to the query sequence, HomoStRscan finds the most similar structure to the input query structure and computes the maximal similarity score (MSS) between the two structures. The homologousRNA structures are then statistically inferred from the MSS distribution computed in the target genome. The method provides a flexible, robust and fine search tool for any homologous structural RNAs.

Motivation: Growing evidence demonstrates that local well-ordered structures are closely correlated with cis-acting elements in the post-transcriptional regulation of gene expression. The prediction of a well-ordered folding sequence (WFS) in genomic sequences is very helpful in the determination of local RNA elements with structure-dependent functions in mRNAs., Results: In this study, the quality of local WFS is assessed by the energy difference (E(diff)) between the free energies of the global minimal structure folded in the segment and its corresponding optimal restrained structure (ORS). The ORS is an optimal structure under the condition in which none of the base-pairs in the global minimal structure is allowed to form. Those WFSs in HIV-1 mRNA, various ferritin mRNAs and genomic sequences containing let-7 RNA gene were searched by a novel method, ed_scan. Our results indicate that the detected WFSs are coincident with known Rev response element in HIV-1 mRNA, iron-responsive elements in ferritin mRNAs and small let-7 RNAs in Caenorhabditis elegans, Caenorhabditis briggsae and Drosophila melanogaster genomic sequences. Statistical significance of the WFS is addressed by a quantitative measure Zscr(e) that is a z-score of E(diff) and extensive random simulations. We suggest that WFSs with high statistical significance have structural roles involving their sequence information., Availability: The source code of ed_scan is available via anonymous ftp as ftp://ftp.ncifcrf.gov/pub/users/shuyun/scan/ed_scan.tar.

Cis-acting elements in post-transcriptional regulation of gene expression are often correlated with distinct local RNA secondary structure. These structures are expected to be significantly more ordered than those anticipated at random because of evolutionary constraints and intrinsic structural properties. In this study, we introduce a computing method to calculate two quantitative measures, NRd and Stscr, for estimating the uniqueness of an RNA secondary structure. NRd is a normalized score based on evaluating how different a natural RNA structure is from those predicted for its randomly shuffled variants. The lower the score NRd the more well ordered is the natural RNA structure. The statistical significance of NRd compared with that computed from structural comparisons among large numbers of randomly permuted sequences is represented by a standardized score, STSCR: We tested the method on the trans-activation response element and Rev response element of HIV-1 mRNA, internal ribosome entry sequence of hepatitis C virus, Tetrahymena thermophila rRNA intron, 100 tRNAs and 14 RNase P RNAs. Our data indicate that functional RNA structures have high Stscr, while other structures have low Stscr. We conclude that RNA functional molecules and/or cis-acting elements with structure dependent functions possess well ordered conformations and they are uniquely folded as measured by this technique.

Herein we provide a new insight into the hydrophobic effect in protein folding. Our proposition explains the molecular basis of cold denaturation, and of intermediate states in heat and their absence in cold denaturation. The exposure of non-polar surface reduces the entropy and enthalpy of the system, at low and at high temperatures. At low temperatures the favorable reduction in enthalpy overcomes the unfavorable reduction in entropy, leading to cold denaturation. At high temperatures, folding/unfolding is a two-step process: in the first, the entropy gain leads to hydrophobic collapse, in the second, the reduction in enthalpy due to protein-protein interactions leads to the native state. The different entropy and enthalpy contributions to the Gibbs energy change at each step at high, and at low, temperatures can be conveniently explained by a two-state model of the water structure. The model provides a clear view of the dominant factors in protein folding and stability. Consequently, it appears to provide a microscopic view of the hydrophobic effect and is consistently linked to macroscopic thermodynamic parameters.

Local folding in mRNAs is closely associated w ith biological functions. In this study, we reveal the whole distribution of local thermodynamic stability in the complete genome of the poliovirus P3/Leon/37 and the single-stranded RNA sequences that corresponds to the nucleotide sequence of the complete genome sequence (1 667 867 bp) of Helicobacter pylori (H. pylori) strain 26695. Local thermodynamic stability in the RNA sequences is measured by two standard z -scores, significance score and stability score. To estimate the distribution of thermodynamic stability, a model based on the non-central Student's t distribution has been developed. Significant patterns of extremes that are either much more stable or unstable than expected by chance are detected. Our results indicate that the highly stable and statistically more significant folding regions are predominantly in non-coding sequences in the two genome sequences. Moreover, the highly unstable folding regions, on the contrary, are predominantly in the protein coding sequences of H. pylori. The observed differences across the complete genomic sequences are statistically very significant by a chi2-test. These extreme patterns may be useful in searching for target sequences for long-chain antisense RNA and for locating potential RNA functional elements involved in the regulation of gene expression including translation, mRNA localization and metabolism., (Copyright 2001 Academic Press.)

There is growing evidence that some enzymes catalyze reactions through the formation of short-strong hydrogen bonds as first suggested by Gerlt and Gassman. Support comes from several experimental and quantum chemical studies that include correlation energies on model systems. In the present study, the process of proton transfer between hydroxyl and imidazole groups, a model of the crucial step in the hydrolysis of RNA by the enzymes of the RNase A family, is investigated at the quantum mechanical level of density functional theory and perturbation theory at the MP2 level. The model focuses on the nature of the formation of a complex between the important residues of the protein and the hydroxyl group of the substrate. We have also investigated different configurations of the ground state that are important in the proton transfer reaction. The nature of bonding between the catalytic unit of the enzyme and the substrate in the model is investigated by Bader's atoms in molecule theory. The contributions of solvation and vibrational energies corresponding to the reactant, the transition state and the product configurations are also evaluated. Furthermore, the effect of protein environment is investigated by considering the catalytic unit surrounded by complete proteins--RNase A and Angiogenin. The results, in general, indicate the formation of a short-strong hydrogen bond and the formation of a low barrier transition state for the proton transfer model of the enzyme.

Here, we depict the anatomy of protein structures in terms of the protein folding process. Via an iterative, top-down dissecting procedure, tertiary structures are spliced down to reveal their anatomy: first, to produce domains (defined by visual three-dimensional inspection criteria); then, hydrophobic folding units (HFU); and, at the end of a multilevel process, a set of building blocks. The resulting anatomy tree organization not only clearly depicts the organization of a one-dimensional polypeptide chain in three-dimensional space but also straightforwardly describes the most likely folding pathway(s). Comparison of the tree with the formation of the hydrophobic folding units through combinatorial assembly of the building blocks illustrates how the chain folds in a sequential or a complex folding pathway. Further, the tree points to the kinetics of the folding, whether the chain is a fast or a slow folder, and the probability of misfolding. Our ability to successfully dissect the protein into an anatomy tree illustrates that protein folding is a hierarchical process and further validates a building blocks protein folding model.

A cDNA (2855 nt) encoding a putative cytotoxic ribonuclease (rapLR1) related to the antitumor protein onconase was cloned from a library derived from the liver of gravid female amphibian Rana pipiens. The cDNA was mainly comprised (83%) of 3' untranslated region (UTR). Secondary structure analysis predicted two unusual folding regions (UFRs) in the RNA 3' UTR. Two of these regions (711-1442 and 1877-2130 nt) contained remarkable, stalk-like, stem-loop structures greater than 38 and 12 standard deviations more stable than by chance, respectively. Secondary structure modeling demonstrated similar structures in the 3' UTRs of other species at low frequencies (0.01-0.3%). The size of the rapLR1 cDNA corresponded to the major hybridizing RNA cross-reactive with a genomic clone encoding onconase (3.6 kb). The transcript was found only in liver mRNA from female frogs. In contrast, immunoreactive onconase protein was detected only in oocytes. Deletion of the 3' UTR facilitated the in vitro translation of the rapLR1 cDNA. Taken together these results suggest that these unusual UFRs may affect mRNA metabolism and/or translation.

We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.

The strong requirement for a small segment of the 5'-proximal coding sequence of hepatitis C virus (HCV) is one of the most remarkable features in the internal initiation of HCV mRNA translation. Phylogenetic analysis and RNA folding indicate a common RNA structure of the 5' untranslated region (UTR) of HCV and the animal pestiviruses, including HCV types 1-11, bovine viral diarrhea (BVDV), border disease virus (BDV) and hog cholera (HoCV). Although the common RNA structure shares similar features to that proposed for the internal ribosome entry sequence (IRES) of picornavirus, phylogenetic evidence suggests four new tertiary interactions between conserved terminal hairpin loops and between the terminal hairpin loop of F2b and the short coding sequence for HCV and pestiviruses. We suggest that the higher-order structures of IRES cis-acting elements for HCV and animal pestivirus are composed of stem-loop structures B-C, domains E-H, stem-loop structure J and four additional tertiary interactions. The common structure of IRES elements for these viruses forms a compact structure by these tertiary interactions and stem stacking. The active structural core is centered in the junction domain of E-H that is also conserved in all members of picornaviruses. Our model suggests that the requirement for a small segment of the 5' coding sequence is to form the distinct tertiary structure that facilitates the cis-acting function of the HCV IRES in the internal initiation of the translational control.

The translational control involving internal ribosome binding occurs in poliovirus (PV), human rhinoviruses (HRV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Internal ribosome binding utilizes cis-acting genetic elements of approximately 450 nucleotides (nt) termed the internal ribosome entry sites (IRES) found in these picornaviral 5'-untranslated region (5'UTR). Although these IRES elements are quite different in their primary sequence, a similar folding structure with a conserved 3' structural core exists in the IRES. Phylogenetic analysis and RNA folding of the 5' UTR of picornaviruses, including PV types 1-3, coxsackievirus types A and B, swine vesicular disease virus, echoviruses, enteroviruses (human and bovine), HRV, HAV, EMCV, mengovirus, Theiler's murine encephalomyelitis viruses, FMDV, and equine rhinoviruses, indicates that the predicted conserved structural core is indeed a general structural feature for all members of the picornavirus family. The evolution of a common structural core likely occurred by the gradual addition or deletion of structural domains and elements to preserve a similar tertiary structure that facilitates the utilization of the IRES in specific host-cell environments.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.

Cap-independent translations of viral RNAs of enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, hepatitis A and C viruses (HAV and HCV), and pestivirus are initiated by the direct binding of 40S ribosomal subunits to a cis-acting genetic element termed the internal ribosome entry site (IRES) or ribosome landing pad (RLP) in the 5' noncoding region (5'NCR). RNA higher ordered structure models for these IRES elements were derived by a combined approach using thermodynamic RNA folding, Monte Carlo simulation, and phylogenetic comparative analysis. The structural differences among the three groups of picornaviruses arise not only from point mutations, but also from the addition or deletion of structural domains. However, a common core can be identified in the proposed structural models of these IRES elements from enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, and HAV. The common structural core identified within the picornavirus IRES is also conserved in the 5'NCR of the divergent viruses, HCV, and pestiviruses. Furthermore, the proposed structural motif shares a structural feature similar to that observed in the catalytic core of the group 1 intron. The conserved structural motif from these divergent sequences that looks like the common core region of group 1 introns is probably a crucial element involved in the IRES-dependent translation.

A statistically significant folding region is identified in the 5' untranslated region (5'-UTR) of hepatitis C virus (HCV), bovine viral diarrhea virus and hog cholera virus. This unusual folding region (UFR) detected in HCV encompasses 199 nucleotides (nt) and coincides with the reported internal ribosome entry site or ribosome landing pad (RLP), as determined by the 5' and 3' deletions [Tsukiyama-Kohara et al., J. Virol. 66 (1992) 1476-1483]. The RNA structure predicted in the UFR of HCV consists of a large stem-loop and a pseudoknot. The proposed structural model is consistent with RNase sensitivity studies [Brown et al., Nucleic Acids Res. 20 (1992) 5041-5045]. Moreover, the structure is highly conserved among these divergent HCV and pestivirus RNAs. The covariation of paired bases in the helical regions offers support for the proposed structural models. The pseudoknot predicted in these UFR shares a similar structural feature to those proposed in the RLP of cardioviruses, aphthoviruses and hepatitis A virus. Based on the common structural motif, a putative base-pairing model between HCV RNA and 18S rRNA, as well as pestiviral RNAs and 18S rRNA are suggested. Intriguingly, the proposed base-pairing models in this study are comparable to those proposed in picornaviruses in terms of their folded shape and location of the predicted complementary sequences between viral RNAs and 18S rRNA. Taken together, we suggest that the common base-pairing model between the UFR detected in the 5'-UTR of pestivirus and HCV and 18S rRNA have a general function in the internal initiation of cap-independent translation.

We have developed a procedure, composed of a set of computer programs, for predicting common RNA structures of homologous sequences. Given a set of homologous RNAs, these programs perform a multiple sequence alignment, generate a list of possible helical stems that are thermodynamically favored in RNA folding from a selected individual sequence, establish a conserved stem list by inspecting the equivalent base pairings and/or conserved helical stems from the derived alignment of homologous RNAs, and build common RNA secondary structures with the maximum scores (i.e., compensatory base changes and number of base pairs, etc.). The approach is a combination of phylogenetic and thermodynamic methods and has been applied to the prediction of common folding structures of the 5' untranslated regions in a number of positive RNA viruses.

We have used molecular modeling techniques to model the RNA tertiary structure of the viral RNA element (referred to as domain II of Rev responsive element, RRE) bound by the Rev protein of HIV. In this study, the initial three-dimensional model was built from its established RNA secondary structure, including three non-Watson-Crick G:G, G:A and G:U base pairs. Molecular dynamics (MD) simulations were performed with hydrated or unhydrated sodium ions. Our results indicate that the non-Watson-Crick base pairs in the simulation with unhydrated sodium ions and water are more stable than those with hydrated sodium ions only. The RNA can maintain its compact double helical structure throughout the course of the MD simulations with water and unhydrated sodium ions, although the non-Watson-Crick base pairs and two bulge loops show much more flexibility and conformational distortion than the classical RNA helical region. The distinct distortion of the sugar-phosphate backbone significantly widens the RNA major groove so that the major groove is readily accessible for hydrogen bonding by specific Rev binding. This model emphasizes the importance of specific hydrogen bonding in the stabilization of the three-dimensional structure of the HIV Rev core binding element, not only between the nucleotide bases, but also among the ribose hydroxyls, phosphate anionic oxygens, base oxygens and nitrogens, and bridging water molecules. Moreover, our results suggest that sodium ions play an important role in the formation of base pairs G:G and G:A of the RRE by a manner similar to the arginine of the Rev-RRE complex.

Infectious bronchitis virus (IBV) mRNA3 encodes three small proteins, 3a, 3b, and 3c, at its 5' end. Recently, it was demonstrated that initiation of protein 3c is dependent on the upstream sequence. Monte Carlo simulations of RNA folding in this tricistronic mRNA3 indicate that a highly significant folding region occurs prior to the initiator AUG of 3c. The unusual folding region (UFR) of 265 nucleotides (nt) contains the coding sequences of proteins 3a and 3b. Details of the structural analyses show that five highly significant RNA stem-loops in the UFR can be modeled into a compact superstructure by the interaction of two predicted pseudoknot structures. The folded superstructure comprising nt 44 to 330, with additional 22 nt downstream from this UFR, is suggested to serve as a ribosome landing pad (or an internal ribosomal entry site) in the cap-independent translation of the 3c of IBV. Intriguingly, the proposed structural motif of this coronavirus shares structural features similar to those proposed in a number of picornavirus mRNAs. Based on the common structural features, a plausible base pairing model between mRNA3 and 18 S rRNA is suggested, which is consistent with a general mechanism for regulation of internal initiation described in many picornaviruses.

Statistical analyses of RNA folding in 5' nontranslated regions (5'NTR) of encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, foot-and-mouth disease virus, and hepatitis A virus indicate that two highly significant folding regions occur in the 5' and 3' portions of the 5'NTR. The conserved tertiary structural elements are predicted in the unusual folding regions (UFR) for these viral RNAs. The theoretical, common structural elements predicted in the 3' parts of the 5'NTR occur in a cis-acting element that is critical for internal ribosome binding. These structural motifs are expected to be highly significant from extensive Monte Carlo simulations. Nucleotides (nt) in the conserved single-stranded polypyrimidine tract for these RNAs are involved in a distinctively tertiary interaction that is located at about 15 nt prior to the initiator AUG. Intriguingly, the proposed common tertiary structure in this study shares a similar structural feature to that proposed in human enteroviruses and rhinoviruses. Based on these common structural features, plausible base pairing models between these viral RNAs and 18 S rRNA are suggested, which are consistent with a general mechanism for regulation of internal initiation of cap-independent translation.

In this paper we present a new method for predicting a set of RNA secondary structures that are thermodynamically favored in RNA folding simulations. This method uses a large number of 'simulated energy rules' (SER) generated by perturbing the free energy parameters derived experimentally within the range of the experimental errors. The structure with the lowest free energy is computed for each SER. Structural comparisons are used to avoid multiple generation of similar structures. Computed structures are evaluated using the energy distribution of the lowest free energy structures derived in the simulation. Predicted be graphically displayed with their occurring frequencies in the simulation by dot-plot representations. On average, about 90% of phylogenetic helixes in the known models of tRNA, Group I self-splicing intron, and Escherichia coli 16 S rRNA, were predicted using the method.

A 50-nucleotide (nt) untranslated region (coding gap sequence) that interrupts the amino acid coding sequence in T4 gene 60, plus an additional 5 nt upstream and another 3 nt downstream from the gap sequence, shows unusual folding patterns according to RNA structure prediction. A predicted highly stable and significant hairpin structure in the 5' half of the gap sequence and a plausible tertiary structural element computed in the 3' part of the gap sequence seem significant by statistical tests on the wild-type (wt) sequence. This feature is absent in insertion, deletion and substitution variants of the gap sequence, in which template activities are markedly lower than that of the wt. The proposed feature is consistent with currently available data showing that the translational bypass of the coding gap is correlated with a stop codon involved in a stem-loop structure folded in the gap sequence. We suggest that the role of this segment in 'ribosomal bypass' of a portion of the mRNA sequence is a property of its special folded structure.

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

The simplest dynamic algorithm for planar RNA folding searches for the maximum number of base pairs. The algorithm uses O(n3) steps. The more general case, where different weights (energies) are assigned to stacked base pairs and to the various types of single-stranded region topologies, requires a considerably longer computation time because of the partial backtracking involved. Limiting the loop size reduces the running time back to O(n3). Reduction in the number of steps in the calculations of the various RNA topologies has recently been suggested, thereby improving the time behavior. Here we show how a "jumping" procedure can be used to speed up the computation, not only for the maximal number of base pairs algorithm, but for the minimal energy algorithm as well.

Here we present the SequenceEditingAligner system for editing multiple, aligned genetic sequences. This is an interactive multi-window color system that displays more than 3500 nucleotides or amino acids. The system handles nucleic acid or protein sequences with or without secondary structure data. More than 300 sequences, each more than 1500 elements in length, may be analyzed together. With the system scientists can classify elements, align sequences, edit them, find consensus patterns, and simultaneously generate oligomer frequency histograms and other statistics.

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.

Current secondary structure prediction computations have a serious drawback. The calculated thermodynamically most stable structure often differs from that observed in solution or in crystal form. In this paper we suggest a way to partially over-come some of these limitations by simulating the RNA folding process and calculating the frequencies of occurrence of the various substructures obtained. The frequently recurring substructures are then selected to construct the secondary structure of the whole RNA. 142 tRNA molecules and an E. coli 16S rRNA molecule have been examined by this method. The percentage of successful prediction of the correct helices are significantly higher than those calculated previously. The secondary structures of intervening sequences (IVSs) excised from human alpha-like globin pre-mRNAs are also computed. Thus, in this method the secondary structures obtained are composed of the statistically more significant substructures. This has also been demonstrated by using randomly shuffled sequences. The secondary structures of each of the randomized sequences are computed and their mean and standard deviations are used in evaluating the significance of the substructures obtained in the folding of the biological sequence. Some potentially appealing structural features aligning adjacent exons for ligation have been found.

An interactive computer system using a dot matrix approach has been developed and used to determine potentially significant features due to distortions in the B-DNA helix as a result of variations of purine and pyrimidine patterns. Sequences were compared using matrices which were generated using the Calladine-Dickerson rules (C.R. Calladine, J. Mol. Biol. 161, 343-352, 1982 and R.E. Dickerson, J. Mol. Biol. 166, 419-441, 1983). Having control over various parameters to enhance different aspects of the visual appearance of these matrices was helpful in discovering patterns that were not known a priori. Specifically, it was found that a pattern of alternating doublets of purines and pyrimidines appear to exist in regulatory regions. This event is shown to be beyond probabilistic expectation.

We have isolated and characterized a segment of the chick alpha 2 collagen gene by screening a library of chick genomic fragments using as hybridization probe an alpha 2 collagen cDNA clone. Several clones were isolated and one of them, lambda gCOL 204, was used for further studies. The DNA of lambda gCOL 204 hybridizes to a unique species of mRNA the size of alpha 2 collagen mRNA. This mRNA can be translated into a unique polypeptide which comigrates in SDS-gel electrophoresis with pro-alpha 2 collagen. Electron microscopic analysis by R-loop technique indicates that lambda gCOL 204 contains 7Kb of the alpha 2 collagen gene. This 7 Kb piece constitutes the 3' end of the gene. The same clone also contains 9 Kb of DNA that is immediately adjacent to the 3' end of the alpha 2 collagen gene. The cloned segment of the alpha 2 collagen gene is interrupted by 8 intervening sequences of various lengths. The coding sequences for collagen in this clone add up to approximately 1,800 bp, which correspond to about 1/3 of alpha 2 collagen mRNA. DNA sequence analysis of a small coding segment of lambda g COL 204 reveals a characteristic collagen type sequence which encodes for an amino acid sequence identical to a sequence found in calf alpha 2 collagen. The sequence of this region of the protein has not yet been determined for the chick alpha 2 collagen.

We report here the nucleotide sequence corresponding to two large regions of the hepatitis A virus (HAV) genome. These comprise a sequence of 3274 bases corresponding to the 5' end of the genome, which includes the putative capsid protein region of this picornavirus, and 1590 bases corresponding to the 3' end of the genome, terminating in a 15-base poly(A) tract. These sequences revealed that HAV had the characteristic genomic organization of picornaviruses: an open reading frame beginning approximately 750 bases from the 5' end of the RNA and a termination codon 60 bases from the 3' poly(A) tract. The predicted amino acid sequences of both regions have been compared to analogous regions previously determined for other picornaviruses. There was sufficient homology to conclude that the 5' region of HAV codes for capsid proteins and that the 3' region codes for an RNA polymerase. However, these regions of HAV were not found to be closely related to analogous regions of poliovirus, encephalomyocarditis virus, and foot and mouth disease virus.

The sizes of the human and subhuman alpha-subunits of the glycoprotein hormones differ by four amino acids (hCG alpha, 92 amino acids; murine, equine, bovine, and ovine alpha, 96 amino acids). The shortening of the human alpha-subunit has been attributed to posttranslational proteolysis. We have recently determined the nucleotide sequences of the mRNAs encoding the precursors of the alpha-subunit of mouse TSH and rat gonadotropins using recombinant DNA techniques. In this report, we have compared these nucleotide sequences and their deduced amino acid sequences with those of the pre- alpha-subunit of hCG (hCG pre-alpha) and the gene encoding the human alpha-subunit. We show that the difference in size of four amino acids between the apoproteins of the murine and human alpha-subunits is a result of a deletion, or insertion, of four codons close to the site of the second intron in the human gene. In addition, the sequence of four codons absent in the mRNA encoding hCG pre-alpha is similar to a region in the 3' end of this intron. These findings indicate that the differences in size of these alpha-subunits originate at the gene level rather than at the posttranslational level at which proteolytic processing occurs.

There has been a rapid increase in the number of available protein sequences derived from gene-sequence information. Computer-based sequence analysis of proteins is gaining in importance as an analytical tool. With the help of these analyses such sequences may be characterized and some insights gained into their probable role in the system. The principles involved in computer-based sequence analysis and some of the methods are discussed.

We have determined the nucleotide sequence of a cloned beta-crystallin cDNA (pM beta Cr1) derived from the 5- to 10-day-old mouse lens and compared its deduced amino acid sequence with the amino acid sequences of the principal beta-crystallin polypeptide (beta Bp) (Driessen, H. P. C., Herbrink, P., Bloemendal, H., and de Jong, W. W. (1980) Exp. Eye Res. 31, 213-216) and a gamma-crystallin polypeptide (gamma II) (Croft, L. R. (1972) Biochem. J. 128, 961-970) of the bovine lens. A previous comparison indicated that bovine beta Bp and gamma II contain internally homologous sequences of amino acids and are evolutionarily related, suggesting an intragenic duplication of a common ancestral gene (Driessen, H. P. C., Herbrink, P., Bleomendal, H., and de Jong, W. W. (1980) Exp. Eye Res. 31, 213-216). When the amino acids were aligned, we calculated a 43% homology between the murine and bovine beta-crystallin polypeptides and a 22% homology between the murine beta-crystallin and bovine gamma-crystallin polypeptides. As for bovine beta Bp and gamma II, there is a striking homology between the amino and carboxyl halves of the murine beta-crystallin polypeptide. The internally homologous amino acids have been preferentially conserved among the three crystallin chains, a fact consistent with the possibility that these lens proteins arose from a common precursor gene that internally duplicated.

We describe a program for the analysis of RNA secondary structure. There are two new features in this program. (i) To get vector speeds on a vector pipeline machine (such as Cray X-MP/24) we have vectorized the secondary structure dynamic algorithm. (ii) The statistical significance of a locally 'optimal' secondary structure is assessed by a Monte Carlo method. The results can be depicted graphically including profiles of the stability of local secondary structures and the distribution of the potentially significant secondary structures in the RNA molecules. Interesting regions where both the potentially significant secondary structures and 'open' structures (single-stranded coils) occur can be identified by the plots mentioned above. Furthermore, the speed of the vectorized code allows repeated Monte Carlo simulations with different overlapping window sizes. Thus, the optimal size of the significant secondary structure occurring in the interesting region can be assessed by repeating the Monte Carlo simulation. The power of the program is demonstrated in the analysis of local secondary structures of human T-cell lymphotrophic virus type III (HIV).

We have determined the nucleotide sequence of a 5.5-kilobase segment of cloned mouse DNA which includes regions encoding two parts of the mouse kappa immunoglobulin gene: the J regions (amino acids 96-108 of the kappa chain) and the C region (residues 109-214). This sequence allows us to rule out interruptions in the germline constant region coding segment as well as the presence of additional functional J genes in the sequenced DNA segment, although two weak homologies to J regions have been found. The complete sequence also allows us to identify a single occurrence of the heptanucleotide palindrome thought to play a role in V/J joining. This palindrome, midway between J and C regions, is the site of aberrant joining in the plasmacytoma MPC11 and may be a target for such aberrant recombination of other kappa genes. In addition, computer analysis of the J sequences suggests that those closest to the C region arose by the most recent duplication event.

The use of modern techniques has led to new insights into the molecular mechanisms of viral pathogenesis. Although the infectious process is quite complex, it is clear that one critical stage, the interaction of viral attachment proteins with cell-surface receptors, often has a major role in determining the pattern of infection. The mammalian reoviruses have served as useful models for understanding the molecular basis of viral pathogenesis. The mammalian reovirus haemagglutinin (sigma 1 protein), which is an outer capsid protein, has been shown to be a major factor in determining virus-host cell interactions. To further our understanding of the structure and function of the haemagglutinin, we have cloned a complementary DNA copy of the reovirus type 3 S1 double-stranded RNA gene which encodes the virus haemagglutinin and have sequenced the DNA complementary to the S1 gene. Analysis of the predicted amino-acid sequence of the virus haemagglutinin has allowed us to determine that the amino-terminal portion contains an alpha-helical coiled-coil structure and that the carboxy-terminal portion contains the receptor-interacting domains. Using this information, we propose here a model of how the reovirus haemagglutinin is attached to the virus particle.

The Mr 19,000 adenovirus glycoproteins coded by the E3 region that is expressed on the cell membrane and presumably binds to HLA class I antigen shows sequence homology to the alpha-chain of human HLA-DR and to the kappa-chain of human IgM. The homology extends to a conserved domain present in all of the major histocompatibility complex antigens, beta 2-microglobulins, and immunoglobulins. Predicted beta-sheet secondary structures are similar between the Mr 19,000 adenovirus protein and these immune system proteins. Evolutionary and functional implications of the homology are discussed.