Current models of rho-dependent transcription termination suggest that RNA polymerase (RNAP) must first synthesize a length of nascent RNA that is bound by rho upstream of the complex; then, rho translocates 5'-> 3' along the RNA until it reaches the transcription elongation complex; the transcribing RNAP is slowed by specific DNA rho termination sequences, thereby allowing rho to “catch up” to the transcribing polymerase at the correct time to properly terminate transcription.

The goal of our studies is to understand the interaction between rho and the transcription complex. To determine if the rho-RNA interactions are necessary for the termination activity of rho, we set out to investigate if rho is capable of termination without a long length of nascent RNA. We synthesized 76 nucleotide DNA oligomers as an artificial transcription bubble. The DNA has 16 complementary bps, followed by 12 non-complementary nucleotides, followed by complementary 48 base pairs containing a rho termination sequence. The 12-nucleotide sequence serves as the bubble. Using T7 RNAP, we synthesized a 12-nucleotide sequence of RNA containing fluorescein-labeled UTPs. This RNA is complementary to the bubble. We purified the labeled RNA and annealed the labeled RNA to the complementary DNA bubble. We are now set to carry out transcription reactions using E. coli RNAP in order to extend the labeled RNA. We will perform single turnover elongation reactions under the same conditions in the presence of rho, in order to test the rho termination activity in the absence of a long strand of nascent RNA.