Structural Energetics of the Adenine Tract from an Intrinsic Transcription Terminator

Intrinsic transcription termination sites generally contain a tract of adenines in the DNA template that yields a tract of uracils at the 3′ end of the nascent RNA. To understand how this base sequence contributes to termination of transcription, we have investigated two nucleic acid structures. The first is the RNA–DNA hybrid that contains the uracil tract 5′-rUUUUUAU-3′ from the tR2 intrinsic terminator of bacteriophage λ. The second is the homologous DNA–DNA duplex that contains the adenine tract 5′-dATAAAAA-3′. This duplex is present at the tR2 site when the DNA is not transcribed. The opening and the stability of each rU-dA/dT-dA base pair in the two structures are characterized by imino proton exchange and nuclear magnetic resonance spectroscopy. The results reveal concerted opening of the central rU-dA base pairs in the RNA–DNA hybrid. Furthermore, the stability profile of the adenine tract in the RNA–DNA hybrid is very different from that of the tract in the template DNA–DNA duplex. In the RNA–DNA hybrid, the stabilities of rU-dA base pairs range from 4.3 to 6.5 kcal/mol (at 10 °C). The sites of lowest stability are identified at the central positions of the tract. In the template DNA–DNA duplex, the dT-dA base pairs are more stable than the corresponding rU-dA base pairs in the hybrid by 0.9 to 4.6 kcal/mol and, in contrast to the RNA–DNA hybrid, the central base pairs have the highest stability. These results suggest that the central rU-dA/dT-dA base pairs in the adenine tract make the largest energetic contributions to transcription termination by promoting both the dissociation of the RNA transcript and the closing of the transcription bubble. The results also suggest that the high stability of dT-dA base pairs in the DNA provides a signal for the pausing of RNA polymerase at the termination site.