Escherichia coli RNA polymerase has subunit composition (alpha)2BetaBetasigma. RNA polymerase alpha subunit carries out three important functions (i) alpha serves as the initiator for RNA polymerase assembly, (ii) alpha participates in promoter recognition by direct sequence-specific protein-DNA interaction, and (iii) alpha is the target for a large set of transcription activator proteins, including catabolite gene activator protein (CAP). Our research project on RNA polymerase alpha subunit is directed at four long-range objectives: (i) determination of the three-dimensional structure of RNA polymerase, (ii) understanding promoter recognition, (iii) understanding transcription initiation, and (iv) understanding transcription activation. Our research project has three specific aims:
Specific Aim 1 : Analysis of domain organization of alpha. Experiments are proposed to dissect a into protease-resistant protein fragments (""""""""domains), to determine the N-terminal and C-terminal boundaries of alpha domains, and to analyze the oligomerization properties, assembly properties, and DNA binding properties of alpha domains.
Specific Aim 2 : Analysis of structure of alpha and alpha domains. Experiments are proposed to prepare gram quantities of alpha and alpha domains, to determine secondary structure compositions by CD spectroscopy, and to determine tertiary structures by multi-dimensional NMR spectroscopy and x-ray crystallography.
Specific Aim 3 : Analysis of DNA binding by alpha. Experiments are proposed to develop preoperative and quantitative assays for alpha-DNA interaction; to determine the """"""""optimal"""""""" DNA-site sequence and minimum DNA-site length for alpha-DNA interaction; to quantify the DNA binding stoichiometry, DNA binding affinity, and DNA binding specificity of 4 alpha-DNA interaction; to identify DNA binding residues by site-specific 5-bromouracil-mediated photocrosslinking, site-specific aldehydic-abasic- site-mediated crosslinking, and alanine-scanning mutagenesis; and to analyze the role of alphaDNA interaction in transcription initiation. The results to be obtained will be directly relevant to understanding regulation of prokaryotic gene expression. Since eukaryotic RNA polymerase subunits share sequence and mechanistic homologies with E. coli RNA polymerase subunits, including a, the results to be obtained also may be relevant to understanding regulation of eukaryotic gene expression.
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