Description
TitleStructural and mechanistic basis of reiterative transcription initiation
Date Created2021
Other Date2021-10 (degree)
Extent1 online resource (xiii, 77 pages) : illustrations
DescriptionIn standard transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a “scrunching” mechanism, in which RNAP remains bound to the promoter, unwinds additional DNA, and pulls the additional unwound DNA past its active center, synthesizing an RNA product having a 5’ sequence complementary to the DNA template. In an alternative pathway of transcription initiation, termed “reiterative transcription initiation,” primarily observed at promoters containing homopolymeric sequences at or near the transcription start site, RNAP binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a mechanism that has not previously been defined--generates an RNA product having a 5’ sequence that contains a variable number of nucleotides not complementary to the DNA template. It has been hypothesized that reiterative transcription initiation may involve “slippage” of the RNA product relative to the DNA template. However, direct evidence for slippage, and information on the relationship between scrunching and possible slippage has not been presented.
In this work, we use x-ray crystallography to define the structures of reiterative transcription initiation complexes with in crystallo RNA synthesis in Thermus thermophilus RNAP RPo crystals. We determined crystal structures of RNAP engaged in standard transcription initiation on a template containing a non homopolymeric sequence and RNAP engaged in reiterative transcription initiation on templates containing template strand GGG and CCC homopolymeric sequences. The complexes in crystal contained 4-5 nt RNA product. The results reveal that RNA extension in reiterative transcription initiation (1) occurs without DNA scrunching, (2) involves a short, 2 bp (post-translocated state) to 3 bp (pre-translocated state) RNA-DNA hybrid, (3) and can involve an RNA product positioned as in standard transcription initiation and a DNA template strand positioned differently from standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with sliding of RNA relative to DNA within a short, 2-3 bp, RNA-DNA hybrid.
We also use cryo-EM to define structures of reiterative transcription initiation complexes synthesizing long, >10 nt, RNA products, seeking to define the path by which such RNA products leave the RNAP active-center cleft. We determined cryo-EM structure of RNAP engaged in reiterative transcription initiation on templates containing template strand CCC homopolymeric sequences. With an 11 nt RNA product, the structure reveals a pathway that can accommodate the long RNA product to exit from, and extend outside, the RNAP. Several conformational changes of RNAP and unique interactions in this RPrtc structure also provide hints on effect regions that may influence the competition between standard transcription and reiterative transcription. Based on the results, we also proposed a mechanism of switching from reiterative transcription initiation to standard transcription initiation.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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