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Enhancement of transcription termination factor rho activity with potassium glutamate

1991; Elsevier BV; Volume: 266; Issue: 16 Linguagem: Inglês

10.1016/s0021-9258(18)99210-0

1083-351X

Lee Zou, J P Richardson,

Bacterial Genetics and Biotechnology

The efficiencies of rho action as a termination factor during transcription in vitro of several DNA templates were determined as a function of the concentration and type of electrolyte ions. The termination efficiencies with lambda-tR1 and the promoter proximal lacZ intragenic terminators were significantly higher with 0.1-0.2 M potassium glutamate as the major electrolyte than with the optimal concentrations of KCl (approximately 0.05 M) or potassium acetate (approximately 0.15 M). Similar high efficiencies were obtained with salts of other acidic amino acids but not with a salt of N-acetylglutamic acid or with a mixture of 0.15 M potassium acetate and 0.15 M glycine, and termination was inhibited completely when 0.12 M KCl was present along with 0.12 M potassium glutamate. The salts that give high termination efficiencies have two properties in common; they consist of anions that are also zwitterions, and they are weak chelators of Mg2+ ions. The increase in termination efficiency with potassium glutamate can be ascribed mainly to a facilitation of the reactions of rho with RNA that are coupled to ATP hydrolysis, as the rate of ATP hydrolysis with isolated transcripts as cofactors was about five times higher with 0.15 M potassium glutamate than with 0.05 M KCl, whereas the rates of chain elongation, the general stability of the transcription complexes, and the binding affinity of rho with the transcripts were all very similar under the two conditions. Further analysis revealed that the activation of ATP hydrolysis is an outcome of a shift in the optimum magnesium salt concentration from 0.5 mM with 0.05 M KCl to 4 mM with 0.15 M potassium glutamate. Since glutamate is a relatively weak counterion for cationic groups in proteins, potassium glutamate can be used at 0.15 M without inhibiting the binding of rho to RNA. At that concentration, it serves to buffer the level of free Mg2+ available to stabilize RNA secondary structures that are known to impede rho action on RNA. The two special properties of glutamate together create conditions that allow rho to terminate transcription in vitro at an efficiency that matches the in vivo efficiency with use of a physiological level of K+ ions.