Overproduction of transcription termination factor Rho in Escherichia coli

A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.     

Free RNA-dependent ATPase activity of transcription termination factor Rho: a model of cyclic dissociation and reassembly of Rho protein

RNA-dependent ATPase activity of Rho + and two mutant proteins Rho15 and Rho301 was studied. It was shown that monomeric Rho forms oligomers in the presence of ATP. This ATP-induced structural change of Rho allows protection of the protein from heat inactivation. Poly(C), which highly activates Rho ATPase, was found to potentiate heat inactivation of Rho301, but no Rho + and Rho15, only under optimal conditions of ATP hydrolysis. It was also shown that Rho301 is defective in interaction with RNA. The molecular model postulating that Rho-catalysed ATP hydrolysis with free RNA involves the cyclic process of protein dissociation and reassembly is postulated.     

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis

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