@misc{oai:ir.soken.ac.jp:00000918, author = {根岸, 智史 and ネギシ, トモフミ and NEGISHI, Tomofumi}, month = {2016-02-17, 2016-02-17}, note = {The RNA polymerase of Escherichia coli plays a key role in transcription of all species of cellular RNA. The control of the activity and specificity is now recognized as a major mechanism of the global regulation of transcription.
　　The RNA polymerase consists of four core subunits (2α, β and β') and one of the multiple molecular species of σ subunit. The assembly of RNA polymerase proceeds sequentially under the order: 2α→α₂→α₂β→α₂ββ' (core enzyme)→ Eσ(holoenzyme). The α subunit consists of 329 amino acid residues and plays essential roles in protein-protein contacts not only for RNA polymerase assembly but also for transcription activation by class-I factors and DNA UP elements. To reveal the structure-function relationship of α subunit, I attempted to reveal the organization of structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. Results indicate that one region, Arg235 - lu244, is highly accessible to both endoproteases. I propose that the α subunit consists of two major structural domains, amino (N) terminal-proximal domain upstream from Arg235 and carboxy (C) terminal-proximal domain downstream from Glu 245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement of its functional map. The N-terminal domain corresponds to the assembly domain of core enzyme and the C-terminal domain corresponds to the transcription activation domain including the contact sites with class-I transcription factors and DNA UP element. Based on detailed analysis of the secondary proteolytic cleavage sites, intra-domain structures are also proposed. The N-terminal domain was cleaved into two subdomain fragments (Na and Nb) between Arg45 and Glu68. The subdomain structure is discussed in relation to the location of subunit-subunit contact sites. On the other hand, the C-terminal proximal domain of 85 amino acids in length (aa residues 245 - 329) was highly resistant to endoproteases. This domain was expected to form a compact structure.
　　Next, I tried to determine the conformation of this C-terminal domain (αCTD) by NMR analysis in collabolation with Prof. Y. Kyogoku and colleagues (Institute for Protein Research, Osaka University). The PCR-amplified fragment of the rpoA, including the coding region for αCTD, was inserted into an expression vector pET-3a to make pETαCTD. The plasmid was transformed into BL21(λDE3) and αCTD was expressed in the transformants. αCTD was purified by ion exchange column chromatography (DEAE-TOYOPEAL) and gel filtration column chromatography (TOYOPEAL-HW55F). The purity of αCTD for 1H NMR analysis was higher than 99% as juged by SDS-PAGE followed by stainning with Coomassie brilliant blue. ¹⁵N-labeled and ¹³C/¹⁵N-double labeled αCTD were expressed in M9 medium containg ¹⁵NH₄Cl (0.05%) and ¹³C-D-glucose (0.1％) as nitrogen and carbon sources. The purification of labeled sample was performed by the column procedures employed for purification of the unlabeled sample. Purity of the sample was more than 99%. The structure of αCTD was determined by multidimentional heteronuclear magnetic resonance spectroscopy in Prof. Kyogoku's laboratory.
　　The NMR analysis revealed that αCTD consists of four helices and two long loops at both termini, together forming a compact and rigid structure. The four helices, helix1 ( Val264 to Leu273), helix2 (Ile278 to Gln283), helix3 (Glu286 to Thr292) and helix 4 (Lys297 to Ser309) are considered to form the hydrophobic core. The helix 1 is perpendicular to the largest helix 4 and thus the N-termini of helix 1 and helix 4 are very close to each other on the tertiary structure. The locations of the contact sites for class-I transcription factors, mapped based on mutant studies, are discussed in relation to the tertiary structure of αCTD. The tertiary structure of αCTD indicated that CRP contact site is located on the helx 1.
　　In order to determine the residue of αCTD to interact with rrnBP1 UP element, the chemical shift perturbation experiments were carried out in Prof. Kyogoku's laboratory by using ¹⁵N-labeled αCTD and the rrnBP1 promoter UP element duplex DNA. Results indicated that the helix 1 and the N-terminal region of helix 4 interact with rrnBP1 promoter UP element. It is worthwhile to note that the tertiary structure of αCTD shows that the relative configuration between helix 1 and helix 4 is similar to the helix-turn-helix motif which is widely observed in DNA-binding proteins.
　　Finally, I tried to confirm the CRP contact site by inhibition assay of lac transcription using four synthetic peptides (A, B, C and D) as inhibitors. These peptides were synthesized with an automated peptide synthesizer by the standard tert-butoxycarbonyl (t-Boc) method using phenyl-acetamidometyl (PAM) resin and purified by μ BONDASPHERE column (reverse phase HPLC). These peptides were designed as to include parts or whole of the CRP contact site (Arg265 to Leu270) and helix1. Peptide C containing both the CRP contact site and the entire helix1 showed the highest activity of inhibition than any of the test peptides. In the case of lack a half of the helix1 (peptide B) or substitution of Ala for Arg265 (peptide D), the level of lac transcription inhibition was half the level of peptide C. Peptide A (tetrapeptide including Arg265) did not inhibit lac transcription. The lac transcription inhibition experiment showed that a peptide including the whole sequence of helix 1 significantly inhibited CRP-RNA polymerase interaction, suggesting that the intact conformation of helix 1 is necessary for effective interaction with CRP. The inhibition test of transcription by synthetic peptides will be used for mapping contact sites with other class-I factors., application/pdf, 総研大甲第235号}, title = {Structure-Function Relationship of the Alpha Subunit of Escherichia coli RNA polymerase}, year = {} }