On the Binding of tRNA to Escherichia coli RNA Polymerase

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Abstract
The fixation of tRNA to E. coli RNA polymerase was investigated. Bound and free tRNA were separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. No differences were detected between the fixation of E. coli .**GRAPHIC**. .**GRAPHIC**. or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and monomer and dimer forms of the core enzyme. In the monomer complex, 1 tRNA is bound/.alpha.2.beta..beta.'' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per .alpha.2.beta..beta.'' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. Binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA exist. The results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.