Mean free energy topology for nucleotide sequences of varying composition based on secondary structure calculations.

The mean free energy generated from the secondary structure of RNA sequences of varying length and composition has been studied by way of probability theory. The expected boundaries or maximal and minimal values of a given distribution are explored and a method for estimating error as a function of the number of shuffled sequences is also examined. For typical nucleotide sequences found in biologically active organisms, the mean free energy, free energy distributions and errors appear to be scalable in terms of a fixed set of algorithm-dependent parameters and the nucleotide composition of the particular sequence under evaluation. In addition, a general semi-analytical formula for predicting the mean free energy is proposed which, at least to first-order approximation, can be used to rapidly predict the mean free energy of any sequence length and composition of RNA. The general methodology appears to be algorithm independent. The results are expected to provide a reference point for certain types of analysis related to structure of RNA or DNA sequences and to assist in measuring the somewhat related matter of complexity in algorithm development. Some related applications are discussed.     

Fluctuation-driven directional flow in biochemical cycle: further study of electric activation of Na,K pumps

Directional flow of information and energies is characteristic of many types of biochemical reactions, for instance, ion transport, energy coupling during ATP synthesis, and muscle contraction. Can a fluctuating force field, or a noise, induce such a directional flux? Previous work has shown that Na,K-ATPase of human erythrocyte can absorb free energy from an externally applied random-telegraph-noise (RTN) electric field to pump Rb+ up its concentration gradient. However, the RTN field used in these experiments was constant in amplitude and would not mimic fluctuating electric fields of a cell membrane. Here we show that electric fields which fluctuate both in life time and in amplitude, and thus, better mimicking the transmembrane electric fields of a cell, can also induce Rb+ pumping by Na,K-ATPase. A Gaussian-RTN-electric field, or a field with amplitude fluctuating according to the Gaussian distribution, with varied standard deviation (sigma), induced active pumping of Rb+ in human erythrocyte, which was completely inhibited by ouabain. Increased values for sigma led to a nonmonotonic reduction in pumping efficiency. A general formula for calculating the ion transport in a biochemical cycle induced by fluctuating electric field has been derived and applied to a simple four-state electroconformational coupling (ECC) model. It was found that the calculated efficiency in the energy coupling decreased with increasing sigma value, and this effect was relatively small and monotonic, whereas experimental data were more complex: monotonic under certain sets of conditions but nonmonotonic under different sets. The agreement in general features but disagreement in some fine features suggest that there are other properties of the electric activation process for Na,K-ATPase that cannot be adequately described by the simple ECC model, and further refinement of the ECC model is required.     

Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.     

In vitro murine leukemia retroviral integration and structure fluctuation of target DNA

Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.     

Theoretical studies on the interaction of proteins and nucleic acids. I. The binding of beta-pleated sheets to A- and B-DNA

A theoretical investigation of the interaction between a beta-ribbon consisting of two glycine hexapeptides and DNA in its A and B conformations is presented. A refined semi-empirical energy formula and a sophisticated energy minimization technique are used to optimize the complex, taking into account the DNA-beta-ribbon interaction, the full flexibility of the oligopeptide chains and of the positions of the DNA screening counterions. A considerable flexibility of the beta-ribbon is demonstrated, which allows the polypeptide fragment to interact comfortably with both forms of DNA considered and with different base-pair sequences. The results are discussed in connection with the general problem of DNA-protein recognition.     

A theoretical investigation of the sequence specificity in the binding of the antitumor drug anthramycin to DNA

A theoretical study is presented concerning DNA-anthramycin adducts. By explicit energy minimisations using a semi-empirical energy formula and an advanced algorithm the structural properties and the energetics of this system are analysed. The results obtained demonstrate that the formation of a covalently bound adduct in which anthramycin is attached to the N2 site of a guanine within a DNA fragment is accompanied by a considerable change in the nucleic acid conformation as confirmed by recent experimental evidence. With the use of the "SIR" methodology for treating DNA flexibility the general features of this change are characterised. The sequence specificity of anthramycin binding is investigated and the important role of sequence dependent nucleic acid flexibility brought to light. This theoretical treatment thus provides new elements for the interpretation of the origins of ligand binding specificities.     

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor sigma(B)

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the sigma(B) stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of sigma(B). B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of sigma(B). Neither the energy-responsive activation of sigma(B) nor stress-dependent chaperone gene induction (a sigma(B)-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of sigma(B) are shown to be active in the RplK(-) strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of sigma(B) and suggest that the ribosomes may themselves be the sensors for stress in this system.     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.     

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.