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Determination of DNA sequence, Pol II catalytic activity and TFIIH subunit Tfb3 functions in transcription start site selection in Saccharomyces cerevisiae

Zhu, Yunye (2022) Determination of DNA sequence, Pol II catalytic activity and TFIIH subunit Tfb3 functions in transcription start site selection in Saccharomyces cerevisiae. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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During initiation of transcription by RNA Polymerase II (Pol II) where and how efficiently transcription initiates is determined by a constellation of inputs including DNA sequence and activities of Pol II and General Transcription Factors (GTFs). Not all promoter positions allow initiation as only subsets of DNA sequences are able to be used as Transcription Start Sites (TSSs). Furthermore, the “promoter scanning” process that determines TSS usage in Saccharomyces cerevisiae is sensitive to alterations in Pol II and GTF factor activity. My dissertation focuses on three determinants of TSS selection: DNA sequence, Pol II catalytic activity and GTF TFIIH subunit Tfb3. To dissect Pol II TSS sequence specificity and how Pol II activity alters it, I develop a massively parallel reporter assay “Pol II MASTER” (Pol II MAssively Systematic Transcript End Readout). Using Pol II MASTER, I measure the efficiency of transcription initiation during promoter scanning by S. cerevisiae Pol II for ~1 million unique TSS sequences. Pol II MASTER not only recapitulates known critical qualities of S. cerevisiae TSS -8, -1, and +1 positions but also demonstrates that surrounding sequences modulate initiation efficiency over a wide range. I discover functional interactions between neighboring sequence positions, indicating that adjacent positions likely function together. These results enable development of a predictable model for initiation efficiency at genomic promoters. I demonstrate that Pol II mutants with altered catalytic activity selectively modulate preference for initiating nucleotide. To determine how Tfb3 functions in promoter scanning and interacts with other initiation factors. I identified two classes of tfb3 alleles that confer polar effects on TSS usage, namely they can shift TSS usage either upstream or downstream. I find that tfb3 allele effects on TSS usage show an intermediate pattern between Pol II efficiency alleles and TFIIH processivity alleles, suggesting Tfb3 might function in TSS selection through both Pol II efficiency and TFIIH processivity. I show primary additive genetic interactions between tfb3 alleles and alleles of TFIIH DNA translocase subunit Ssl2 or initiation cofactor Sub1, suggesting Tfb3 functions in regulating TFIIH’s processivity.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Zhu, Yunyeyunye_zhu@pitt.eduyuz1660000-0001-9503-7882
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKaplan, Craig D.craig.kaplan@pitt.edu0000-0002-7518-695X
Committee MemberHainer, Sarah
Committee MemberBrodsky, Jeffrey
Committee MemberRebeiz,
Committee MemberCarvunis,
Date: 6 June 2022
Date Type: Publication
Defense Date: 7 December 2021
Approval Date: 6 June 2022
Submission Date: 4 February 2022
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 229
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Pol II transcription, TSS selection, DNA sequence, catalytic activity, TFIIH, Tfb3
Date Deposited: 06 Jun 2022 15:54
Last Modified: 06 Jun 2023 05:15


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