Structural and Functional Analysis of UBF & Fish UBF Gene Identification

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Project title

Code/計畫編號

NSC83-0209-B019-005

Translated Name/計畫中文名

UBF蛋白結構與功能分析及魚類UBF基因鑑認

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=99812

Year

1994

Start date/計畫起

01-02-1993

Expected Completion/計畫迄

01-07-1994

Bugetid/研究經費

555千元

ResearchField/研究領域

生物科學

本研究工作依原擬計畫分二階段執行,目前已順利完成第一階段之進度,茲將其所獲之初步結果略述如后。本計畫之第一階段研究目標在於對兩棲類RNA polymerase I之轉錄因子UBF其辨認DNA及蛋白耦合之結構與功能之分析。結果:(1)順利將兩棲類UBF之主要DNA辨識功能區(DNA binding domain)-HMG box I及蛋白耦合功能區(dimerization domain)轉殖入E. coli表現及純化。(2)初步測試具有良好DNA辨識及附合能力與蛋白耦合能力。(3)已利用遺傳工程方法著手進行各種胺基酸轉換之工作,藉以探測各重要胺基酸之結構功能。本計畫預期於第二階段完成後,將對UBF之DNA辨識與耦合等之結構機制將有較完整之認識。UBF is an general RNA polymerase I-specific transcription factor which assist other essential factors to assemble stable initiation complex in the promoter of ribosomal genes. We have shown that UBF recognize cross-over DNA structure by its multiple DNA binding domain motifs, named HMG box. In this study, we expressed the major DNA binding motif, HMG box-1, by E. coli overexpression system. We demonstrated that the recombinant peptide bound cross-over DNA. By making series of deletion from both ends, we could define the minimum DNA binding activity within the central 50 residues of this motif. We are making various point-mutations on the conserved amino acid residues and analyzing their DNA binding activities. In addition, we also expressed the N-terminal protein dimerization domain. Coexisting with cluster of basic amino acid residues, this peptide filled in entire double-stranded DNA and formed unique ladders in gel shift assay. We are making series of deletion from C-terminus and internal deletion to define its functional dimerization domain. In addition, we will make various point mutations in the amphipathic region to replace the hydrophobic residues and characterize the function of amphipathic region in protein homodimer formation.

Keyword(s)

RNA聚合酶
轉錄因子
魚
基因分析
RNA polymerase
Transcription factor
Fish
Gene analysis

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Structural and Functional Analysis of UBF & Fish UBF Gene Identification

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