자료유형  

 학위논문

Control Number  

0017162129

International Standard Book Number  

9798382761381

Dewey Decimal Classification Number  

574

Main Entry-Personal Name  

Bushhouse, David Z.

Publication, Distribution, etc. (Imprint  

[S.l.] : Northwestern University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

301 p.

General Note  

Source: Dissertations Abstracts International, Volume: 85-11, Section: B.

General Note  

Advisor: Lucks, Julius B.

Dissertation Note  

Thesis (Ph.D.)--Northwestern University, 2024.

Summary, Etc.  

요약All organisms adapt to changes in their environment, which requires mechanisms to sense the outside world and alter gene expression in response. Transcription, the enzymatic synthesis of RNA from a DNA template, is a highly regulated process that enables organisms to express different genes at different times at different amplitudes. Transcription is subject to many gene regulatory control mechanisms to ensure that expression of each transcript is appropriate for a given cellular state. For example, a suite of conditional attenuators such as RNA thermometers and riboswitches control transcription termination. Riboswitches are an ancient class of cis- regulatory RNA gene-regulatory elements found across all domains of life. These elements take advantage of the ability of RNA to form intricate structures during transcription to sense intracellular concentrations of their cognate ligands, and alter gene expression in response.A long-standing and intriguing question about riboswitch biology is how transcriptional riboswitches can make fast folding decisions on the timescale of active transcription. To date a number of transcriptional riboswitch folding pathways have been deciphered. Collectively, these studies are revealing that transcriptional riboswitches navigate a complex decision landscape that pushes them to one of two final structural states based on the conditional execution of an internal strand displacement process. These bifurcating folding pathways must maintain a delicate balance in order to maintain robust dynamic range, raising the intriguing question of whether transcriptional riboswitches tune their function by manipulating the favorability of strand displacement. Using the Clostridium beijerinckii pfl ZTP riboswitch as a model, we performed fine-grained mutational analysis of the strand displacement process that governs the bifurcation of this riboswitch's folding pathway. These results illustrate that the dynamic range of this riboswitch can be finely tuned over an order of magnitude from 2.4-34-fold by mutating the invading strand to contain kinetic barriers that slow down strand displacement (e.g. mismatches, deletions, bulges). We also investigate the accessory regions of the Cbe pfl riboswitch, identifying key roles for the leader sequence and linker region in controlling riboswitch function. Interestingly, we identify a role for RNAP pausing in controlling ZTP riboswitch dynamic range, but only in the context of purine starvation.Another long-standing question about riboswitch biology is how transcriptional riboswitches tune their sensitivity to ligand. Understanding how riboswitch sensitivity is controlled is critical to understanding how highly conserved aptamer domains are deployed in a variety of contexts with different sensitivity demands. Here we uncover new roles by which RNA folding dynamics control riboswitch sensitivity in cells. By investigating the Clostridium beijerinckii pfl ZTP riboswitch, we identify multiple mechanistic routes of altering expression platform sequence and structure to slow RNA folding, all of which enhance riboswitch sensitivity. Applying these methods to riboswitches with diverse aptamer architectures that regulate transcription and translation with ON and OFF logic demonstrates the generality of our findings, indicating that any riboswitch that operates in a kinetic regime can be sensitized by slowing expression platform folding. Comparison of the most sensitized versions of these switches to equilibrium aptamer:ligand dissociation constants suggests a limit to the sensitivities achievable by kinetic RNA switches. Our results add to the growing suite of knowledge and approaches that can be used to rationally program cotranscriptional RNA folding for biotechnology applications, and suggest general RNA folding principles for understanding dynamic RNA systems in other areas of biology. Careful analysis of ZTP riboswitch expression platform sequences reveal a novel anti- termination mechanism used across bacteria. We find that the presence of nascent RNA structures within the 5' upstream context of a terminator can slow or occlude its formation in the RNAP exit channel, leading to weaker termination due to a 'tug-of-war' (TOW) between the upstream RNA structure and the terminator.

Subject Added Entry-Topical Term  

Biology.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Biophysics.

Subject Added Entry-Topical Term  

Genetics.

Index Term-Uncontrolled  

Biosensing

Index Term-Uncontrolled  

Riboswitch

Index Term-Uncontrolled  

RNA

Index Term-Uncontrolled  

RNA engineering

Index Term-Uncontrolled  

RNA folding

Index Term-Uncontrolled  

RNA structure

Added Entry-Corporate Name  

Northwestern University Interdepartmental Biological Sciences (IBiS) Graduate Program

Host Item Entry  

Dissertations Abstracts International. 85-11B.

Electronic Location and Access  

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Control Number  

joongbu:658561