Site-Resolved Structural Energetics of the T7 Concatemer Junction

Abstract

The concatemer junction is a conserved sequence of 8 bp, which is strategically located at the junction between the head-to-tail repeats of genomic DNA in T7 and related bacteriophages. The RNA polymerase pauses at this site to recruit the machinery necessary for cleavage of the concatemer into single genome DNA. During pausing, the transcription bubble collapses and the transcription RNA–DNA hybrid is shortened to only 3 bp. This work addresses the question of the role of the nucleic acid components of the transcription elongation complex in this collapse of the transcription bubble. The nucleic acid structures investigated are the DNA–DNA duplex structure present at the concatemer junction when the DNA is not transcribed and the RNA–DNA hybrid formed when the concatemer junction is transcribed. The structural energetics of each base pair in the two structures is characterized using imino proton exchange and nuclear magnetic resonance spectroscopy. The results show that 5 bp in the DNA–DNA duplex at the concatemer junction site are significantly more stable than the corresponding base pairs in the RNA–DNA hybrid that forms when the site is transcribed. Because of their energetic preference for the DNA–DNA duplex, these 5 bp favor the collapse of the transcription bubble. Four of the 5 bp with enhanced stability in the DNA–DNA duplex are located in the downstream half of the concatemer junction site. This location suggests that only after the entire concatemer junction is transcribed can the RNA–DNA hybrid accumulate sufficient structural destabilization to trigger the dissociation of the RNA and the switch of the DNA template strand from the hybrid structure to the DNA–DNA double-helical structure