Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo.     

Structure of a C-rich strand fragment of the human centromeric satellite III: a pH-dependent intercalation topology

Repetitive DNA sequences may adopt unusual pairing arrangements. At acid to neutral pH, cytidine-rich DNA oligodeoxynucleotides can form the i-motif structure in which two parallel-stranded duplexes with C.C(+) pairs are intercalated head-to-tail. The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytidine tracts, the nucleotide sequences between them, and the experimental conditions.We have found that a natural fragment of the human centromeric satellite III, d(CCATTCCATTCCTTTCC), can form two monomeric i-motif structures that differ in their intercalation topology and that are favored at pH values higher (the eta-form) and lower (the lambda-form) than 4.6. The change in intercalation may be related to adenine protonation in the loops.We studied the uridine derivative methylated on the first cytidine base, d(5mCCATTCCAUTCCUTTCC), whose proton spectrum is better resolved. The intercalation topologies are (C7.C17)/(5mC1.C11)/(C6.C16)/(C2.C12) for form lambda and (5mC1.C11)/(C7.C17)/(C2.C12)/(C6.C16) for form eta. We have solved the structure of the eta-form, and we present a model for the lambda-form. The switch from eta to lambda involves disruption of the i-motif. In both forms, the central AUT linker crosses the wide groove, and the first and the third linkers loop across the minor grooves. The i-motif core is extended in the eta-form by the inter-loop reverse Watson-Crick A3.U13 pair, whose dissociation constant is around 10(-2) at 0 degrees C, and in the lambda-form by the interloop T5.T15 pair.In contrast, d(5mCCATTCCTTACCTTTCC) folds into a pH-independent structure that has the same intercalation topology as the lambda-form. The i-motif core is extended below by the interloop T5.T15 pair and closed on top by the T8.A10 pair.Thus, the C-rich strand of the human satellite III tandem repeats, like the G-rich strand, can fold into various compact structures. The relevance of these features to centromeric function remains unknown.     

分子内分子伴侣--Pro肽在蛋白质折叠中的作用

在体内,许多蛋白质,如很多胞外蛋白酶、某些多肽激素等都以含前导肽的前体形式合成,前导肽在蛋白质折叠中具有分子伴侣的功能。为了与一般意义上的分子伴侣相区别,人们将对蛋白质折叠有帮助的前导肽称为分子内分子伴侣,分子内分子伴侣帮助蛋白质在折叠过程中克服高的能量障碍,某些蛋白质的分子内分子伴侣甚至促进其在氧化性折叠中二硫键的正确配对。     

Long telomeric C-rich 5'-tails in human replicating cells

Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3'-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3'-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5'-tail, the incompletely replicated template of the leading strand. We examined the presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3'-overhang, the 5'-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5' C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening.     

Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor

A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive alpha-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the alpha-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.     

Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand

As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC). A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band. The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment. Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p. These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers. The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded. We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.     

Intramolecular chaperone: the role of the pro-peptide in protein folding.

Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding.     

Trinucleotide repeats associated with human disease.

Triplet repeat expansion diseases (TREDs) are characterized by the coincidence of disease manifestation with amplification of d(CAG. CTG), d(CGG.CCG) or d(GAA.TTC) repeats contained within specific genes. Amplification of triplet repeats continues in offspring of affected individuals, which generally results in progressive severity of the disease and/or an earlier age of onset, phenomena clinically referred to as 'anticipation'. Recent biophysical and biochemical studies reveal that five of the six [d(CGG)n, d(CCG)n, (CAG)n, d(CTG)n and d(GAA)n] complementary sequences that are associated with human disease form stable hairpin structures. Although the triplet repeat sequences d(GAC)n and d(GTC)n also form hairpins, repeats of the double-stranded forms of these sequences are conspicuously absent from DNA sequence databases and are not anticipated to be associated with human disease. With the exception of d(GAG)n and d(GTG)n, the remaining triplet repeat sequences are unlikely to form hairpin structures at physiological salt and temperature. The details of hairpin structures containing trinucleotide repeats are summarized and discussed with respect to potential mechanisms of triplet repeat expansion and d(CGG.CCG) n methylation/demethylation.     

Rate-temperature relationships in lambda-repressor fragment lambda 6-85 folding

Two classes of lambda(6-85) mutants (those richer in alanine, and those richer in glycine) have very similar slopes in an Arrhenius plot of the unfolding rates but very different temperature dependencies of the folding rates. Temperature-dependent interactions (e.g., hydrophobicity) play a large role in the initial stages of folding but not in the initial stages of unfolding of lambda(6-85). Placement of the transition state in terms of its surface exposure and entropy shows that at least two reaction coordinates are required to describe folding of all mutants over the full temperature range. The unusual Arrhenius plots of the very fastest mutant provide an additional kinetic signature for downhill folding.     

Protein folding and function: the N-terminal fragment in adenylate kinase

Three-dimensional protein folds range from simple to highly complex architectures. In complex folds, some building block fragments are more important for correct protein folding than others. Such fragments are typically buried in the protein core and mediate interactions between other fragments. Here we present an automated, surface area-based algorithm that is able to indicate which, among all local elements of the structure, is critical for the formation of the native fold, and apply it to structurally well-characterized proteins. In particular, we focus on adenylate kinase. The fragment containing the phosphate binding, P-loop (the "giant anion hole") flanked by a beta-strand and an alpha-helix near the N-terminus, is identified as a critical building block. This building block shows a high degree of sequence and structural conservation in all adenylate kinases. The results of our molecular dynamics simulations are consistent with this identification. In its absence, the protein flips to a stable, non-native state. In this misfolded conformation, the other local elements of the structure are in their native-like conformations; however, their association is non-native. Furthermore, this element is critically important for the function of the enzyme, coupling folding, and function.     

G+C-rich tract in 5' end of human introns.

Analysis of an artificial neural network trained to classify DNA as coding or non-coding revealed compositional differences between sequence parts translated into protein and those that were not. The 5' end of human introns was found to have a base composition that was non-random to an extent matching the non-randomness in the 3' end that contains the polypyrimidine tract. The prevailing nucleotides in the initial 50 nucleotides of human introns are guanine and cytosine, the trinucleotide GGG was found to occur almost four times as frequently as it would in sequences with a uniform distribution of the nucleotides. The initial part of terminal exons and their associated terminal introns were shown to have a very special base composition deviating strongly from the normal picture in other exons and introns.     

Alu repeats in the human genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long (LINEs) and short (SINEs) interspersed elements. Alu elements are each a dimer of similar, but not identical, fragments of total size about 300 bp, and originate from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3'-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats comprise more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity, and trapping in the ribosome

Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Analysis of three restriction fragment length polymorphisms in the human type II procollagen gene.

Cloned genomic DNA sequences corresponding to various regions of the human type II procollagen gene were used to analyze the DNA from 78 normal volunteers. Southern hybridization experiments detected polymorphic HindIII, BamHI, and EcoRI sites. The presence of the polymorphic HindIII site results in a 7.0-kilobase (kb) band, and the absence of this site results in a 14.0-kb band. When present, the BamH1 polymorphic site yields a 4.8-kb band, and when absent, yields a 7.2-kb band. The presence of the EcoRI polymorphic site results in a 3.7-kb band, and its absence results in a 7.0-kb band. Each polymorphic site was mapped. Analyses of the data demonstrated that the sites are present in overall gene frequencies of .39 for HindIII, .04 for BamHI, and .02 for EcoRI. Gene frequencies of the polymorphic sites were also studied with respect to race. The polymorphic sites are present in a Hardy-Weinberg distribution in the study population. Study of an extended family demonstrated that the segregation of the HindIII polymorphic site is consistent with Mendelian inheritance.     

Asexual genetic variability inAgavaceae determined with inverse sequence-tagged repeats and amplification fragment length polymorphism analysis

Agaves are succulent monocot plants rich in fibers, sugars and other important compounds. They are also valued as ornamental plants and for their ability to grow in poor soils. In the present study, inverse sequence-tagged repeats (ISTR) and amplified fragment length polymorphism (AFLP) analysis were used to study genetic diversity in differentAgavaceae plant samples. Comparison of the banding patterns between the mother plant and rhizome-derived daughter plants showed that genetic variability is generated during asexual reproduction in these species. Phylogenetic relationships amongAgave species were obtained using unweighted pair-group method, arithmetic average (UPGMA) analysis. Genetic diversity through asexual propagation allows for genetic selection and improvement within these asexually propagated plants.     

Pushing the detection limits: the evanescent field in surface plasmon resonance and analyte-induced folding observation of long human telomeric repeats

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface. In our proof-of-principle experiment we could observe the direct folding of long, human telomeric repeats induced by the small analyte potassium using surface plasmon resonance spectroscopy. This work demonstrates the feasibility of new evanescent field-based biosensors that can specifically observe small molecule interactions.     

The role of alphoid higher order repeats (HORs) in the centromere folding

Understanding the folding of centromere DNA in the maximally condensed methaphase chromosome remains a basic challenge in cell biology. We propose here a set of structural models with a graphical presentation of alphoid higher order repeat (HOR) distribution in the centromere folding, based on the assumption of encryption key for microtubule-centromere interaction which arises from chromosome-specific crystal-like structure of HORs. Specific HOR leads to a characteristic geometrical pattern which may be responsible for individual microtubule to recognize a specific structure of centromere in each chromosome.