NMR Studies of the GCN4 Transcription Factor and Hox DNA Consensus Sequences- [electronic resource]
NMR Studies of the GCN4 Transcription Factor and Hox DNA Consensus Sequences- [electronic resource]
- 자료유형
- 학위논문파일 국외
- 최종처리일시
- 20240214101705
- ISBN
- 9798380130202
- DDC
- 574.191
- 서명/저자
- NMR Studies of the GCN4 Transcription Factor and Hox DNA Consensus Sequences - [electronic resource]
- 발행사항
- [S.l.]: : Columbia University., 2023
- 발행사항
- Ann Arbor : : ProQuest Dissertations & Theses,, 2023
- 형태사항
- 1 online resource(143 p.)
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-02, Section: B.
- 주기사항
- Advisor: Palmer, Arthur.
- 학위논문주기
- Thesis (Ph.D.)--Columbia University, 2023.
- 사용제한주기
- This item must not be sold to any third party vendors.
- 초록/해제
- 요약The conversion of genetic information into functional RNA and protein is of fundamental importance to all known life forms. In cellular organisms, this hinges on the interaction of double stranded DNA and the transcription factor class of proteins. Substantial progress in the fields of biochemistry and genomics have made the identification of transcription factor binding sites and the resultant change in transcriptional output relatively routine. However, fully understanding this central life process requires knowing not only where transcription factors bind DNA, but why and how. These questions are approached here using solution state NMR spectroscopy and the statistical technique of bootstrap aggregation in order to: i) glean biologically relevant insights into the dynamics of the GCN4 transcription factor from NMR relaxation experiments; ii) examine the influence of electrostatics on the structure of GCN4 in the absence of DNA; iii) analyze the conformational state of several Hox transcription factor DNA binding sites.NMR spectroscopy capitalizes on connections between electromagnetism and the quantum mechanical property of nuclear spin angular momentum to study the structure of molecules. Application of NMR relaxation experiments provides further information on molecular structure and dynamics. When performed in solution, the data generated by this technique occurs in conditions more similar to those found within a cell than other approaches used in structural biology.However, the biological relevance of any insights derived from solution state NMR relaxation experiments depends on the application of an appropriate model for nuclear spin relaxation. Typically, this involves applying a statistical test to select the best model from among several candidates in the model-free formalism. Chapter 3 uses 15N relaxation data collected on the basic leucine zipper (bZip) domain of the GCN4 transcription factor to detail the potential problems and model selection errors that arise from this approach, and presents the alternative method of bootstrap aggregation. Applying this statistical technique allowed for the generation of multimodel inferences about the internal motions and rigidity of the basic region of GCN4, enhancing the likelihood of their biological relevance.The results presented in Chapter 3 further confirmed the presence of nascent helices in the generally disordered basic region of the GCN4 bZip domain. Interestingly, when complexed with appropriate DNA substrate, this region assumes a fully α-helical conformation. A long standing hypothesis assumes the inability of the basic region to form an α-helix in the absence of DNA arises, in part, due to repulsion between its charged amino acids. This hypothesis is tested in Chapter 4 using NMR relaxation experiments performed in solutions containing either increased or decreased concentrations of salt. Surprisingly, screening the electrostatic repulsion between charged residues using higher levels of salt had no discernible effect on the structure or dynamics of the basic region.Chapter 5 examines the other side of the interaction between DNA and transcription factors. Here, previous work performed with the Hox family of transcription factors indicated the conformational state of DNA has an important role in enhancing the specificity with which Hox proteins bind certain sequences. In particular, the geometry of the DNA minor groove strongly influences the recruitment of appropriate Hox transcription factors. This relationship is examined using solution state NMR to study four Hox DNA binding sequences. The binding affinity between each of these sequences and the Hox protein AbdB was previously shown to correlate with the native unbound state of the DNA. The two sequences predicted to have native minor groove widths similar to those of the bound DNA had higher affinity for AbdB than those that deformed upon binding. Though mixed, the results of NMR experiments generally support the predicted structures, particularly for the high affinity sequences, indicating a single pronounced narrowing of the minor groove. Taken together, the results presented here illustrate the complex interactions underpinning the appropriate binding of DNA and transcription factors. It further highlights the need to study the structure and dynamics of both DNA and protein, as well as that of the bound complex, in order to fully understand how and why specific sequences are bound in response to stimuli.
- 일반주제명
- Biophysics.
- 일반주제명
- Molecular biology.
- 일반주제명
- Biochemistry.
- 키워드
- Proteins
- 키워드
- Electrostatics
- 기타저자
- Columbia University Cellular Molecular and Biomedical Studies
- 기본자료저록
- Dissertations Abstracts International. 85-02B.
- 기본자료저록
- Dissertation Abstract International
- 전자적 위치 및 접속
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