TY - JOUR TI - Sequence-specific interactions between RNA polymerase and the core recognition element DO - https://doi.org/doi:10.7282/T38055RD PY - 2017 AB - In bacteria, the flow of biological information from DNA to RNA is carried out by a single enzyme called RNA polymerase (RNAP). Bacterial RNAP is composed of a multi-subunit catalytic core and a dissociable subunit called sigma factor. Since the discovery of sigma factor in 1969, the prevailing view has been that the RNAP core enzyme requires binding to sigma for sequence-specific transcription, because the RNAP core does not contain the determinants for sequence-specific core promoter recognition and DNA unwinding. It has also been assumed that sequence-specific RNAP-DNA interactions are mainly limited to transcription initiation, as sigma could dissociate from the RNAP core after the initiation stage. These two paradigmatic assumptions have been challenged by recent structural evidence from our lab, which indicates in the initiation complex, the RNAP core directly interacts with the non-template strand segment of the transcription bubble corresponding to positions -4 to +2, and that the interaction with this element is sequence-specific at least at one of its positions. This element has been termed the “core recognition element,” CRE. This thesis addresses three major topics regarding CRE: sequence-specificity, recognition mechanism, and the functional roles. In chapter 1, using equilibrium binding and dissociation kinetics studies, I demonstrate that the RNAP core shows sequence-specificity at 3-out-of-6 CRE positions (the consensus sequence is T₋₄ n₋₃ n₋₂ n₋₁ T₊₁ G₊₂). I also determine that RNAP amino acid βR371 mediates specificity at CRE position -4, βW183 mediates specificity at CRE position +1, and βR151, βD446, or βR451 mediates specificity at CRE position +2. In subsequent chapters, I use the RNAP derivative containing the βD446A substitution as a reagent to assess the functional significance of RNAP-CRE⁺²ᴳ interactions on transcription initiation and elongation. In chapters 2, 3 and 4, using a combination of next-generation sequencing approaches and biophysical and biochemical assays, I show that sequence-specific RNAP-GCRE interactions play functional roles in three key stages of transcription initiation: promoting DNA unwinding at a consensus GCRE sequence, favoring start-site selection at positions upstream of a consensus GCRE sequence, and reducing the probability of abortive transcript release at positions upstream of a consensus GCRE sequence. In chapter 5, using biochemical assays and mNET-seq, I show that sequence-specific RNAP-GCRE interaction occurs in and plays functional roles in key stages of transcription elongation through the E. coli genome: favoring pause-read-through at positions upstream of consensus GCRE sequence and favoring post-translocated states at positions upstream of consensus GCRE sequence. In chapter 6, using a promoter-independent transcription assay, I show that RNAP-GCRE interaction occurs in, and plays functional roles in all three domains of life: bacteria, archaea and eukaryote. In chapter 7, using genome-wide next-generation sequencing approaches, I show that the RNAP core can perform sequence-specific transcription in the absence of sigma factor in a manner that correlates with the presence of an AT-rich region followed by a TG-motif. In the final chapter, I summarize how my work revealed previously undocumented regulatory events in transcription initiation and elongation. Based on my findings, I describe two implications of this thesis for future consideration: a scenario describing what the architecture of primordial promoter sequences might have looked like and a mechanism for antibiotic-tolerant persistence state. KW - Biochemistry KW - RNA polymerases LA - eng ER -