Two Approaches to the Study of the Mechanism of the Transition Process from Initiation to Elongation in T7 Rna Polymerase

• Main Author: Redhu, Shiv K
• Format: Others
• Published: ScholarWorks@UMass Amherst 2008
• Subjects: Biochemistry
• Online Access: https://scholarworks.umass.edu/theses/173; https://scholarworks.umass.edu/cgi/viewcontent.cgi?article=1248&context=theses

Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. However, the mechanism of the instability of abortive cycling in RNA polymerase is not been well understood. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies that reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes, we now propose that collapse contributes to the release of abortive products in the presence of all four NTPs. Specifically, we propose that stabilizing initially transcribing complexes against downstream bubble collapse will allow these complexes to escape better to full run elongation. This study will provide important mechanistic insight, but will also be valuable for the production of high quantities of RNA from highly abortive DNA sequences. We have recently proposed a new model for the transition from initiation to elongation in T7 RNA polymerase. DNA-to-DNA FRET measurements have allowed mapping the changes that occur before promoter release. To observe the movements of the rotating domain after promoter release requires protein labeling. We are developing a new technique of affinity directed site specific labeling of the protein. We have also begun to explore an intein mediated strategy for direct labeling of T7 RNA polymerase.