Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies.
In this paper we examine the binding of Escherichia coli transcription termination factor rho to single-stranded RNA. Random polyribonucleotide copolymers containing low ratios of the fluorescent base 1,N6-ethenoadenosine have been synthesized using polynucleotide phosphorylase. Binding of rho to these polynucleotides elicits a significant increase in fluorescence, thus allowing either the direct monitoring of the titration of these polynucleotides with rho or measurement of the competitive displacement of the protein from these probes with other nucleic acids, even in the presence of biologically significant concentrations of ATP. By these techniques, it is shown that the binding site size (n) of rho protein to polynucleotides is 13(+/- 1) nucleotide residues per rho monomer (or 78(+/- 6) nucleotide residues per rho hexamer). Binding constants (K) and co-operativity parameters (omega) for the binding of rho to these polynucleotides have been measured as a function of nucleotide composition and of salt concentration. The results show that the affinity of rho for cytosine residues is quite strong and salt concentration independent, whilst binding to uridine residues is somewhat weaker and very salt concentration dependent. Poly(rC) and poly(dC) bind to rho competitively and with equal affinity and site size, although poly(rC) is the strongest cofactor for activating rho-dependent ATPase and poly(dC) has no ATPase cofactor activity at all. It is also shown that ATP (or ADP or ATP-gamma-S) binding does not change the binding site size of rho on RNA nor decrease its affinity for RNA binding. Circular dichroism measurements of rho binding to phage R17 RNA suggest that the affinity (K omega) of rho for RNA may be increased by ATP. The possible significance of these results for models of rho-dependent transcription termination is discussed in the companion paper.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000495:Allosteric Site; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D012313:RNA / Q000378:metabolism*; D012234:Rho Factor / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012234:Rho Factor; D014157:Transcription Factors; D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate