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theses

In bacteria, initial transcription occurs through a scrunching mechanism, where RNA Polymerase (RNAP) remains stationary on promoter DNA, unwinding and pulling downstream DNA into itself and past its active center. The incorporation of additional nucleotides into the active center cleft leads to expansion of the single-stranded transcription bubble, with the accumulated DNA on each strand proposed to be accommodated as single-stranded bulges in the unwound region. The location of these single-strand DNA bulges as they proceed to increase in size during initial transcription has not been fully elucidated.
In this work, we have used single-molecule fluorescence resonance energy transfer (FRET) to detect and analyze the path of nontemplate strand (NT) DNA at multiple increased levels of scrunched DNA. We have also defined the positions of scrunched NT DNA during scrunching using FRET-derived distance restraint docking onto structural models of RNAP.
We have prepared static initial transcription complexes (ITCs) with iteratively increasing scrunched states in the NT strand: RPo, 2 nucleotides (nt) scrunched, 4 nt scrunched, 6 nt scrunched, and 8 nt scrunched. The complexes were shown to be properly formed and fully functional in transcription.
In this work, we have demonstrated the specific incorporation of fluorescent probes at intended labeling sites in both RNAP and NT strand DNA, and have demonstrated the resulting DNA:RNAP derivative complexes are functionally functional. We have determined probe-probe single-molecule FRET distances for each labeled DNA:RNAP combination as part of each static scrunched NT strand complex, totaling 160 unique combinations.
By combining the FRET results with distance restraint docking methodology, we have established models of NT strand DNA in context of RPo, 2 nt scrunched, 4 nt scrunched, 6 nt scrunched, and 8 nt scrunched. The RPo models were compared to solved RPo crystal structures to validate our methodology. Additionally, these models were formed for scrunched complexes containing either a consensus (DSC) discriminator sequence or anticonsensus (aDSC) discriminator sequence. Models were analyzed to both determine the path of NT strand DNA as it proceeds through higher levels of scrunching and to compare the different pathways seen between DSC and aDSC complexes.
Our work showed that the models fit well with crystals structures of RPo. It also showed that scrunched NT strand DNA can be accommodated within the active center cleft up to at least 6-8 nt of scrunched DNA. Comparison of structures demonstrated that aDSC complexes have more flexibility in their possible locations within the active center cleft during scrunching and they may exit from the active center cleft at an earlier scrunched state.

ETD_9235

Ph.D.

Includes bibliographical references

by Adam Hasemeyer

doi:10.7282/t3-pqdp-x863

ETD doctoral

The author owns the copyright to this work.