Strong magnesium binding sites in yeast phenylalanine transfer RNA.

To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op....Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m2G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it links the dihydrouridine arm with the extra (V) loop.     

Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.     

RNA polymerase binding sites on the broad host range plasmid RP4

Binding sites of Escherichia coli RNA polymerase on RP4 plasmid DNA were determined electron microscopically. Comparison of the RNA polymerase binding map and the genetic map of RP4 revealed several strong binding sites outside the well-known RP4 genes. RNA polymerase binding sites for the three antibiotic resistance genes were also detected. Two binding sites were observed for the tra-1 region, whereas the tra-2 and tra-3 regions showed no prominent affinity for RNA polymerase. The genomic regions for the replication origins, oriV (for vegetative replication) and oriT (for transfer replication, equivalent to rlx), both exhibited strong binding to RNA polymerase, as did genomic regions which code for trans-acting replication functions (trfA and trfB).