Alanine is helix-stabilizing in both template-nucleated and standard peptide helices

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451–454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249–4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template–helix junction. A model in which the helix propensities of residues at the template–peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template–peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.

Binding of RNA template to a complex of HIV-1 reverse transcriptase/primer/template

HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.

From residue matching patterns to protein folding topographies: General model and bovine pancreatic trypsin inhibitor

A coarse-grained model for protein-folding dynamics is introduced based on a discretized representation of torsional modes. The model, based on the Ramachandran map of the local torsional potential surface and the class (hydrophobic/polar/neutral) of each residue, recognizes patterns of both torsional conformations and hydrophobic-polar contacts, with tolerance for imperfect patterns. It incorporates empirical rates for formation of secondary and tertiary structure. The method yields a topological representation of the evolving local torsional configuration of the folding protein, modulo the basins of the Ramachandran map. The folding process is modeled as a sequence of transitions from one contact pattern to another, as the torsional patterns evolve. We test the model by applying it to the folding process of bovine pancreatic trypsin inhibitor, obtaining a kinetic description of the transitions between the contact patterns visited by the protein along the dominant folding pathway. The kinetics and detailed balance make it possible to invert the result to obtain a coarse topographic description of the potential energy surface along the dominant folding pathway, in effect to go backward or forward between a topological representation of the chain conformation and a topographical description of the potential energy surface governing the folding process. As a result, the strong structure-seeking character of bovine pancreatic trypsin inhibitor and the principal features of its folding pathway are reproduced in a reasonably quantitative way.

Sources of selection bias in evaluating social programs: An interpretation of conventional measures and evidence on the effectiveness of matching as a program evaluation method

This paper decomposes the conventional measure of selection bias in observational studies into three components. The first two components are due to differences in the distributions of characteristics between participant and nonparticipant (comparison) group members: the first arises from differences in the supports, and the second from differences in densities over the region of common support. The third component arises from selection bias precisely defined. Using data from a recent social experiment, we find that the component due to selection bias, precisely defined, is smaller than the first two components. However, selection bias still represents a substantial fraction of the experimental impact estimate. The empirical performance of matching methods of program evaluation is also examined. We find that matching based on the propensity score eliminates some but not all of the measured selection bias, with the remaining bias still a substantial fraction of the estimated impact. We find that the support of the distribution of propensity scores for the comparison group is typically only a small portion of the support for the participant group. For values outside the common support, it is impossible to reliably estimate the effect of program participation using matching methods. If the impact of participation depends on the propensity score, as we find in our data, the failure of the common support condition severely limits matching compared with random assignment as an evaluation estimator.

Chemical phenotype matching between a plant and its insect herbivore

Two potential outcomes of a coevolutionary interaction are an escalating arms race and stable cycling. The general expectation has been that arms races predominate in cases of polygenic inheritance of resistance traits and permanent cycling predominates in cases in which resistance is controlled by major genes. In the interaction between Depressaria pastinacella, the parsnip webworm, and Pastinaca sativa, the wild parsnip, traits for plant resistance to insect herbivory (production of defensive furanocoumarins) as well as traits for herbivore “virulence” (ability to metabolize furanocoumarins) are characterized by continuous heritable variation. Furanocoumarin production in plants and rates of metabolism in insects were compared among four midwestern populations; these traits then were classified into four clusters describing multitrait phenotypes occurring in all or most of the populations. When the frequency of plant phenotypes belonging to each of the clusters is compared with the frequency of the insect phenotypes in each of the clusters across populations, a remarkable degree of frequency matching is revealed in three of the populations. That frequencies of phenotypes vary among populations is consistent with the fact that spatial variation occurs in the temporal cycling of phenotypes; such processes contribute in generating a geographic mosaic in this coevolutionary interaction on the landscape scale. Comparisons of contemporary plant phenotype distributions with phenotypes of herbarium specimens collected 9–125 years ago from across a similar latitudinal gradient, however, suggest that for at least one resistance trait—sphondin concentration—interactions with webworms have led to escalatory change.

Temperature, template topology, and factor requirements of archaeal transcription

Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100°C, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the β-γ phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.

Double-level “orthogonal” dynamic combinatorial libraries on transition metal template

Dynamic combinatorial libraries are mixtures of compounds that exist in a dynamic equilibrium and can be driven to compositional self adaptation via selective binding of a specific assembly of certain components to a molecular target. We present here an extension of this initial concept to dynamic libraries that consists of two levels, the first formed by the coordination of terpyridine-based ligands to the transition metal template, and the second, by the imine formation with the aldehyde substituents on the terpyridine moieties. Dialdehyde 7 has been synthesized, converted into a variety of ligands, oxime ethers L11–L33 and acyl hydrazones L44–L77, and subsequently into corresponding cobalt complexes. A typical complex, Co(L22)22+ is shown to engage in rapid exchange with a competing ligand L11 and with another complex, Co(L22)22+ in 30% acetonitrile/water at pH 7.0 and 25°C. The exchange in the corresponding Co(III) complexes is shown to be much slower. Imine exchange in the acyl hydrazone complexes (L44–L77) is strongly controlled by pH and temperature. The two types of exchange, ligand and imine, can thus be used as independent equilibrium processes controlled by different types of external intervention, i.e., via oxidation/reduction of the metal template and/or change in the pH/temperature of the medium. The resulting double-level dynamic libraries are therefore named orthogonal, in similarity with the orthogonal protecting groups in organic synthesis. Sample libraries of this type have been synthesized and showed the complete expected set of components in electrospray ionization MS.

Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies

Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H·C base pairs are thermodynamically more stable than other H·N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k2app for pairing with standard dNTPs by factors of 10–1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H·C >> H·T > H·A > H·G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2–20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Translesional synthesis on a DNA template containing N2-methyl-2′-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N2-methyl-2′-deoxyguanosine (N2-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N2-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N2-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N2-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3′-terminus of a dT:N2-Me-dG pair was only 2.1 times lower than from a dC:N2-Me-dG pair. We conclude that N2-Me-dG is a miscoding lesion capable of generating G→A transition mutations.

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.

A complexity measure for selective matching of signals by the brain.

We have previously derived a theoretical measure of neural complexity (CN) in an attempt to characterize functional connectivity in the brain. CN measures the amount and heterogeneity of statistical correlations within a neural system in terms of the mutual information between subsets of its units. CN was initially used to characterize the functional connectivity of a neural system isolated from the environment. In the present paper, we introduce a related statistical measure, matching complexity (CM), which reflects the change in CN that occurs after a neural system receives signals from the environment. CM measures how well the ensemble of intrinsic correlations within a neural system fits the statistical structure of the sensory input. We show that CM is low when the intrinsic connectivity of a simulated cortical area is randomly organized. Conversely, CM is high when the intrinsic connectivity is modified so as to differentially amplify those intrinsic correlations that happen to be enhanced by sensory input. When the input is represented by an individual stimulus, a positive value of CM indicates that the limited mutual information between sensory sheets sampling the stimulus and the rest of the brain triggers a large increase in the mutual information between many functionally specialized subsets within the brain. In this way, a complex brain can deal with context and go "beyond the information given."

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.