The long-range goal of this project is to define the interactions in the transcription complex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV- 1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Although significant progress has been made in understanding pausing, termination, and the regulatory proteins that control these events, two alternative models remain possible. In one view, called the allosteric model, pause signals, termination signals, and regulatory proteins primarily affect the conformation of RNA polymerase. In the other, these signals and proteins primarily affect translocation of a relatively rigid RNA polymerase on the RNA and DNA chains (the rigid-body model). Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems. A combination of biochemical, genetic, and biophysical approaches will be used to distinguish the allosteric and rigid-body models of transcriptional regulation, and to characterize the mechanisms of pausing, termination, and regulatory proteins that control them.
Specific aims will be to (i) characterize interactions of RNA polymerase's flap-tip helix with RNA, NusA, and sigma-70, and test how these interactions affect catalysis in the active site; (ii) determine the location of the RNA 3 about end in paused and nonpaused transcription elongation complexes; (iii) determine the kinetic mechanisms of elongation, pausing, and termination; (iv) map interactions between RNA polymerase and pause and terminator hairpins; and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation.
|Ray-Soni, Ananya; Mooney, Rachel A; Landick, Robert (2017) Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact. Proc Natl Acad Sci U S A 114:E9233-E9242|
|Harwig, Alex; Landick, Robert; Berkhout, Ben (2017) The Battle of RNA Synthesis: Virus versus Host. Viruses 9:|
|Mishanina, Tatiana V; Palo, Michael Z; Nayak, Dhananjaya et al. (2017) Trigger loop of RNA polymerase is a positional, not acid-base, catalyst for both transcription and proofreading. Proc Natl Acad Sci U S A 114:E5103-E5112|
|Zhang, Jinwei; Landick, Robert (2016) A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure. Trends Biochem Sci 41:293-310|
|Ray-Soni, Ananya; Bellecourt, Michael J; Landick, Robert (2016) Mechanisms of Bacterial Transcription Termination: All Good Things Must End. Annu Rev Biochem 85:319-47|
|Bae, Brian; Nayak, Dhananjaya; Ray, Ananya et al. (2015) CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition. Proc Natl Acad Sci U S A 112:E4178-87|
|Czyz, Agata; Mooney, Rachel A; Iaconi, Ala et al. (2014) Mycobacterial RNA polymerase requires a U-tract at intrinsic terminators and is aided by NusG at suboptimal terminators. MBio 5:e00931|
|Hein, Pyae P; Kolb, Kellie E; Windgassen, Tricia et al. (2014) RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement. Nat Struct Mol Biol 21:794-802|
|Larson, Joshua; Kirk, Matt; Drier, Eric A et al. (2014) Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9:2317-28|
|Zhang, Yan; Mooney, Rachel A; Grass, Jeffrey A et al. (2014) DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest. Mol Cell 53:766-78|
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