Cooperative Thermal Denaturation of the Assembly Origin Region of TMV RNA

Abstract

The assembly origin (AO) region of the tobacco mosaic virus RNA melts in an unusually narrow(2.5°C) temperature range. In an 0.01 M phosphate buffer the melting temperature of AO was found to be 41.5°C. This value corresponds to the regions with the most stable secondary/tertiary structure of the whole TMV RNA molecule. It is assumed that the AO region has a specific tertiary structure, which is maintained by the long-range interactions as well as by interactions of the pseudoknot type.     

Detection of periodic patterns in RNA sequences: the first encapsidated region of the TMV RNA

A method is developed to study the periodic properties of nucleotide sequences allowing the favoured pattern of the repeating unit, as well as the length and localization of this periodic segment to be determined simultaneously. The degree of periodicity is evaluated calculating the probabilities for random occurrence of the maximal deviations of the nucleotide composition in each phase, making use of the binomial formula.The nucleotide sequence of the tobacco mosaic virus (TMV) RNA responsible for recognition of the homologous protein (“assembly origin”, AO) (Zimmern & Butler, 1977) was investigated in order to find periodic regions of primary structure which might be essential in the recognition process. As a result the most periodic segments of the AO consisting of 31 and 17 nucleotides corresponding to the schemes GAU or GA1 have been found. However, the periodicities in these regions do not exceed that expected for random sequences. It can be considered as an evidence that in addition to peculiarities of primary structure, some other features such as RNA secondary or tertiary structure are essential in this interaction.For comparison the nucleotide sequences of the other fragments of TMV RNA as well as MS2 RNA, TYMV RNA, 16S rRNA and phage fd DNA were investigated by the same method.     

Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3' non-coding region

Summary The cloned cDNA derived from the 3 end of cowpea strain (Cc) RNA of tobacco mosaic virus (TMV) has been sequenced. Substantial sequence information of 1,060 nucleotides from the 3 end of the RNA reveals some interesting features: (1) the coat protein cistron corresponds to residues 210–701 from the 3 end. Some errors in the amino acid sequence previously reported have been corrected and the revised total length of the coat protein is 162 amino acid residues. The capping site of the coat protein mRNA is at residue 711 from the 3 end of genome RNA. (2) The assembly origin of reconstitution is positioned within the coat protein cistron at residue 369–461 which can be formed into a highly base-paired hairpin loop structure. The sequence, GAXGUUG, in the loop region and a triplet-repeated purine base tract surrounding the loop are found. These structural features are common to assembly origins of both Cc and vulgare strains. (3) We find the sequence highly homologous to, but distinct from, the genuine assembly origin. It will be called the pseudo-assembly origin, which is located in the corresponding region to the assembly origin of the vulgare strain, outside the coat protein cistron. There is also the sequence, GAXGUUG, in the middle of the region. (4) In the 5 flanking region of the coat protein cistron, a long reading frame, probably of 30 K protein, is found. The coding region is terminated in the coat protein cistron and thus the 30 K protein and the coat protein cistrons overlap. (5) The 3 non-coding region is 209 residues long and can be folded into a possible tRNA-like structure. Surprisingly, we find that the 3 terminal sequence of Cc RNA is not very similar to that of vulgare RNA but extensively homologous to that of turnip yellow mosaic virus (TYMV) RNA.     

A theory of aggregation in the thermal denaturation region of multistrand biopolymers

A theory explicitly allowing the possibility of aggregation of multistrand biopolymers is proposed. It is found that the same secondary bonds responsible for stabilizing the native structure at low temperature will promote aggregation in the thermal denaturation region for sufficiently long chains. A requirement for both open and zippered regions dictates that the aggregation region does not extend far below T_m. However, its width, or extension on the high-temperature side of T_m, is a strongly increasing function of chain length and also of the cooperativity parameter. The present theoretical results obtained for DNA and collagen with almost no adjustable parameters are in good qualitative agreement with a number of previously poorly understood experimental observations. The significance of such a spontaneous aggregation phenomenon for genetic recombination is noted.     

An extended secondary structure model for the TMV assembly origin, and its correlation with protection studies and an assembly defective mutant

Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.     

Cooperative assembly of EBNA1 on the Epstein-Barr virus latent origin of replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates DNA replication by binding to multiple copies of its 18-bp recognition sequence present in the Epstein-Barr virus latent origin of DNA replication, oriP. Using electrophoretic mobility shift assays, we have localized the minimal DNA binding domain of EBNA1 to between amino acids 470 and 607. We have also demonstrated that EBNA1 assembles cooperatively on the dyad symmetry subelement of oriP and that this cooperative interaction is mediated by residues within the minimal DNA binding and dimerization domain of EBNA1.     

Cooperative denaturation kinetics of homogeneous polymers

The kinetics of denaturation of a homogeneous, helical biopolymer with nearest neighbor interactions are described, using a kinetic Ising model in which the configuration of its neighbors dictates the transition probability for a single residue in the chain. The actual kinetics that are simulated using Monte Carlo techniques are compared with the results of analytical kinetic equations for the fraction of helix, <s>, generated using the mean-field approximation. This mean-field rate equation is expanded as a hierarchy of terms that characterize the nature of rate constants for interacting systems. The first term in the expansion is first order in <s> and varies linearly with the interaction energy. Subsequent rate terms involve higher powers of <s> and demonstrate the need for nonlinear equations in systems with larger interaction energies. Both the simulations and the mean-field approximation show an intrinsic induction period for the single-step kinetic process. They also yield an apparent first-order rate constant that changes as the reaction proceeds. However, only the simulated kinetics yield ordered regions of chain and a nonzero, nearest-neighbor correlation function.     

Force-induced denaturation of RNA

We quantitatively describe an RNA molecule under the influence of an external force exerted at its two ends as in a typical single-molecule experiment. Our calculation incorporates the interactions between nucleotides by using the experimentally determined free energy rules for RNA secondary structure and models the polymeric properties of the exterior single-stranded regions explicitly as elastic freely jointed chains. We find that despite complicated secondary structures, force-extension curves are typically smooth in quasi-equilibrium. We identify and characterize two sequence/structure-dependent mechanisms that, in addition to the sequence-independent entropic elasticity of the exterior single-stranded regions, are responsible for the smoothness. These involve compensation between different structural elements on which the external force acts simultaneously and contribution of suboptimal structures, respectively. We estimate how many features a force-extension curve recorded in nonequilibrium, where the pulling proceeds faster than rearrangements in the secondary structure of the molecule, could show in principle. Our software is available to the public through an "RNA-pulling server."     

Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.     

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.     

High-resolution analyzer of thermal denaturation

Details are given for the construction of a high-resolution denaturation analyzer for nucleic acid-containing macromolecules. The system contains the following new components: Peltier elements, guided by a linear resistance thermometer for temperature control, electronic microstirrer for quick thermal equilibration within the sample cell, long-term differentiator circuit for converting the absorption function to the first derivative of the rate of change with respect to time.Highly polymerized salmon DNA was melted in standard saline-citrate and showed a hyperchromicity of 45%. The melting velocity passed through a single sharp maximum coinciding with the T_m at 87°C.Ribosomes from Ehrlich asccites tumor cells were thermally denatured in distilled water and showed a 20% hyperchromicity and an overall T_m of 72°C. The denaturation profile was very complex, indicating a sequence of conformational events partially resolved by the instrument.     

Studies on the thermal denaturation of nucleohistones

The thermal denaturation profiles of nucleohistone from calf thymus, sea urchin sperm and sea cucumber male gonad, are studied and compared under a variety of conditions. These include melting in the presence of either one of the following agents: urea, methanol, divalent cations or excess histones. The influence of ionic strength, pH, formaldehyde treatment and partial denaturation is also studied. Particular attention is given to the factors which influence the bimodal appearance of the profiles. The melting curves of the three materials used are qualitatively similar under all conditions, although they show quantitative differences. The histone:DNA ratio appears to be the most important parameter to define the denaturation properties of a given nucleohistone preparation. It is shown that redistribution of histones may determine the melting profile, since during denaturation histones can migrate from locally denatured regions towards those regions which contain native DNA. It is also shown that there are regions of phosphate negative charges of DNA not protected by histone. These regions can be protected against denaturation either by additional histones or by certain divalent cations. The results are interpreted in terms of the various models possible for the distribution of histones on DNA in native nucleohistone. Their biological significance is also discussed.     

Solid-phase synthesis and thermal denaturation study of cyclic PNAs targeting the HIV-1 TAR RNA loop

Cyclic PNAs targeting the HIV-1 TAR RNA loop have been synthesized following a convenient solid-phase strategy which allows on-resin cyclisation. UV-monitored thermal denaturation studies demonstrate that these cyclic PNAs are able to strongly interact with their TAR RNA target, very likely through the formation of a six-base pair stable complex, involving the TAR RNA loop.     

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.     

The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly

Upon mixing purified TMV RNA with limited amounts of viral coat protein in the form of the disk aggregate, a unique region of the whole RNA becomes protected from nuclease digestion. The protected RNA consists of fragments up to 500 nucleotides long in varying yields, which are found in nucleoprotein particles having a protein-nucleic acid ratio similar to the mature virus. The protected RNA, when reextracted, is able to rebind to coat protein disks rapidly, quantitatively and with high affinity, becoming once more RNAase-resistant in the process. Small aggregates of TMV protein (A protein) are inactive in formation of the nuclease-resistant complexes. On the basis of this evidence, we identify the isolated RNA fragments as portions of TMV RNA containing the origin or initiation site for in vitro reassembly, which have been protected from digestion by incorporation into assembly nucleation complexes.The yield, but not the length distribution, of the protected RNA pieces is found to double upon increasing the protein added from 1–2 disk-equivalents of protein per RNA molecule. This implies that the formation of the nucleation complexes may involve a highly cooperative initial addition of protein.     

Dissociative mechanism of F-actin thermal denaturation

We have applied differential scanning calorimetry to investigate thermal unfolding of F-actin. It has been shown that the thermal stability of F-actin strongly depends on ADP concentration. The transition temperature, T(m), increases with increasing ADP concentration up to 1 mM. The T(m) value also depends on the concentration of F-actin: it increases by almost 3 degrees C as the F-actin concentration is increased from 0.5 to 2.0 mg/ml. Similar dependence of the T(m) value on protein concentration was demonstrated for F-actin stabilized by phalloidin, whereas it was much less pronounced in the presence of AlF4(-). However, T(m) was independent of protein concentration in the case of monomeric G-actin. The results suggest that at least two reversible stages precede irreversible thermal denaturation of F-actin; one of them is dissociation of ADP from actin subunits, and another is dissociation of subunits from the ends of actin filaments. The model explains why unfolding of F-actin depends on both ADP and protein concentration.