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Mechanism of the transition process from initiation to elongation in T7 RNA polymerase
Transcription by DNA-dependent RNA polymerases is characterized by at least two major phases. At initiation, an RNA polymerase recognizes a specific promoter sequence and begins de novo RNA synthesis. During elongation, the RNA polymerase releases the promoter contacts and processively extends the RNA chain in a sequence-independent manner. A third phase, abortive cycling, accompanies the transition from initiation to elongation and is characterized by premature release of short transcripts 2-8 bases in length. In the current study, we focus on different aspects of the transition from initiation to elongation in an ideal model system: bacteriophage T7 RNA polymerase. First, a relatively complete structural model is proposed to elucidate the key features of the transition in a sequential three-step manner, by providing possible intermediate coordinates that bridge the known initiation and elongation complex crystal structures. Second, we focus on major factors that relate to the stability of the abortive complex. Building on previous studies, new results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a DNA construct with weakened promoter binding shows less abortive cycling, confirming that retention of promoter contacts contributes to the release of abortive products. Third, a mechanistic model describing the late stages of the transition process is proposed, based on experimental data. Beyond translocational position +8, two energetically coupled events, promoter release and initial bubble collapse, cooperatively facilitate initial RNA displacement at translocational positions +9-10. Proper displacement of the 5' end of the RNA into the newly formed RNA exit channel ensures that the complex enters the elongation phase. RNA in the complexes that do not have proper initial RNA displacement can only be extended for a few additional bases, resulting in the release of dead end products (mostly 11-13mer). Finally, efficient RNA chain elongation is achieved in a nucleic acid scaffold-derived approach, bypassing the process of de novo initiation either in the presence or the absence of a promoter, suggesting that elongation mode transcription may be achieved from this alternative approach.
Gong, Peng, "Mechanism of the transition process from initiation to elongation in T7 RNA polymerase" (2006). Doctoral Dissertations Available from Proquest. AAI3215895.