Frank B. Howard

Helix formation between polyribonucleotides and purines, purine nucleosides and nucleotides. II

A number of monomeric nucleic acid components react with polynucleotides to form well-defined complexes having helical structures similar to those formed between corresponding pairs of high polymers. The monomer can be a purine or a purine nucleoside or nucleotide, but no pyrimidine derivative has yet been found to form such complexes. The phenomenon has been investigated by infrared spectroscopy and optical rotation and by use of an enzyme of known sensitivity to secondary structure. Base-pairing specificity of interaction is observed, and the components react in definite stoichiometric ratios. The dependence of the stabilities of some of the complexes upon concentration of reactants, concentration of salt, and upon temperature has been determined.

Helix formation between polyribonucleotides and purine nucleosides: III. The effects of purine nucleosides in cell-free amino acid-incorporating systems

The preceding paper in this series demonstrated that 2-aminoadenosine and adenosine form regular ordered structures with poly U or poly UC. The extent of interaction is dependent upon temperature and upon the concentration of the reactants. In the present paper we report that complementary ribosides influence the capacity of poly U or poly UC to direct amino acids into protein in cell-free systems from rat liver and Escherichia coli. Polyphenylalanine synthesis directed by poly U is inhibited in both systems. The incorporation of amino acids directed by poly UC is inhibited by 2-aminoadenosine in the system from rat liver but is stimulated in the system from E. coli. Both the inhibition and the enhancement of the capacity of the polymer to serve as messenger BNA depend upon temperature and upon the concentration of riboside, suggesting that both effects are due to the formation of ordered structure. 2-Aminoadenosine brings about a small but temperature-dependent inhibition of the incorporation of amino acids directed by natural endogenous messenger KNA in rat liver polysomes. 2-Aminoadenosine decreases the rate of degradation of poly U in the complete incorporating system from liver. The nucleoside also interferes with the binding of poly U to ribosomes. These opposing influences offer possible explanations for the observed effects of complementary nucleosides on polyribonucleotide-directed incorporation of amino acid into protein.

EVIDENCE FOR A+(ANTI)-G(SYN)MISMATCHED BASE-PAIRING IN D-GGTAAGCGTACC

Two‐dimensional NMR spectroscopy has been used to study the structure and hydrogen bonding scheme of A:G mismatched base pairing in d‐GGTAAGCGTACC at pH 5.8. Under the conditions of our study, the molecule forms a B‐DNA helix, with the mismatched bases in the A+(anti)‐G(syn) conformation. The adenosine exists in the protonated form. The NOESY spectrum in 90% H2O + 10% 2H2O has been used to assign all observable imino and amino protons including those involved in the A+(anti)‐G(syn) base pair. Both the proton donors in the A:G mismatched inter‐base hydrogen bonding are situated on adenosine.

Lipid Vesicle Aggregation Induced by Cooling

Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 °C to 5 °C) and about 2 micron vesicles of DSPC (from 20 °C to 15 °C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 °C to 5 °C failed to increase aggregation within 24 hours at Mg++/DPPS ratios that begin to initiate aggregation and fusion.

Consequences of Substituting 2NH2A for a in Synthetic DNA’S

Chemical and spectroscopic consequences of replacing A with 2NH2A have been examined in a variety of synthetic DNA’s. This substitution, which permits formation of a third hydrogen bond in AT pairs, increases the stability of these pairs. The Tm elevation, however, is much smaller in the deoxy (∆Tm 12–15°) than in the ribo series (∆Tm 27–33°). Sequence effects appear to be small. CD spectra of all helices having the 2NH2A substitution have a relatively strong extremum at 286 to 298 nm. This band is positive for B-form helices (deoxy-deoxy pairs in low salt) and negative for A-form helices (ribo-ribo and deoxy-ribo pairs). These results are consistent with the unusual CD spectrum of S-2L DNA (Kirnos et al.). This natural DNA has all A’s replaced by 2NH2A and positive CD bands at 290 nm and 265 nm. We assign the band at ~290 nm in these helices to the B2u transition of 2NH2A, displaced to longer wavelength by exciton splitting, and suggest that it is relatively unperturbed by transitions of other bases. Alternating (d2NH2A-dT)n undergoes a cooperative transition to an altered conformation in the presence of 4M NaC1 or 2 x 10−4M hexammine cobalt. CD, IR, and 31P NMR experiments reveal similarities to the behavior of (dG-dC)n as well as some differences. The results are consistent with a Z conformation for the high salt form but do not establish it. The alternating polymers (d2NH2A-dC)n•(dG-dT)n and (d2NH2A-dC)n•(dI-dT)n were also observed with CD. The former did not undergo a discrete transition in high salt. The latter did undergo a transition, but the structural nature of the change is not clear.