Physiochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase.

In a medium containing 10mM Tris, pH 8, 10 mM MG++, 50 mM K+ and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 240 degrees at 37 degrees C. In this medium the total unwinding of the DNA increases linearly with the molar ratio of polymerase to DNA. The number of binding sites at which unwinding can occur is very large. If the K+ concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 140 degrees at 37 degrees C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA is strongly temperature dependent, and decreases with decreasing temperature.     

Subunits of yeast RNA polymerase I involved in interactions with DNA and nucleotides.

Ovine mammary gland mRNAs were translated in a wheat-germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on α-lactalbumin isolated by immunoprecipitation from the mixture of radiolabelled lactoproteins showed the occurrence of an hydrophobic 19 residues long amino terminal extension. The pre-protein represents the primary translation product since the amino terminal methionyl residue was found to be donated by initiator $\text{Met-tRNA}{}_{\mathrm{i}}{}^{\mathrm{Met}}$. Comparison of the signals of ovine α-lactalbumin and hen's egg white lysozyme, two homologous proteins which are thought to be derived from a common ancestor, suggests that the signal region has evolved at least as rapidly as the remaining part of the polypeptide chain.     

Equilibrium measurements of cognate and noncognate interactions between aminoacyl transfer RNA synthetases and transfer RNA.

The interaction of Escherichia coli isoleucyl-tRNA synthetase with its cognate and five noncognate tRNAs, and of yeast valyl-tRNA synthetase with its cognate and four noncognate tRNAs, has been measured directly by fluorescence quenching. The cognate associations are strongest (association constant of 10(8) M-1 or more at pH 5.5, 17 degrees). A wide variation is found in the strengths of the noncognate interactions; these have association constants smaller than that of these cognate association by a factor of less than 10 to over 10(4), depending on the enzyme-t-RNA pair. A more detailed study of the cognate isoleucyl-tRNA synthetase-tRNAIle association suggests that the strength of the interaction is markedly sensitive to a pH-dependent transition in the enzyme centered at pH 6 on the other hand, Mg2+-induced structural changes in tRNAIle at 17 degrees in low salt do not greatly affect the availability of the nucleic acid's receptor sites for enzyme...     

RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA

Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10(2)-10(3)-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10(2)-10(3)-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n - 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.