PMC:4502370 / 34726-38669 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"Structure-guided alanine scan identifies amino acids essential for rpo26Δ complementation\nThe crystal structure of Rpo26 in the context of RNA polymerase II highlights a network of intramolecular side chain contacts entailing salt bridge, hydrogen bonding, and π-cation interactions (Figure 6A). Here, we performed a structure-guided alanine scan of eight residues that comprise this network: Arg79, Glu89, Arg97, Glu124, Arg135, Arg136, Asp145, and Glu150. The alanine mutations were introduced into the biologically active RPO26-(78-155) gene on 2-µ plasmids under the control of the TPI1 promoter and tested for complementation of rpo26∆ by plasmid shuffle. Four of the alanine mutations were lethal: E89A, E124A, R135A, and R136A. Three of the alanine mutants—R79A, D145A and E150A—were viable and grew as well as “wild-type” RPO26-(78-155) at 18°, 25°, 30°, and 37° (Figure 6B). R97A cells grew at 25° and 30° but displayed cs and ts defects, whereby they failed to grow at 18° and grew slowly at 37°, as gauged by colony size (Figure 6B). We interpret the mutational data in light of the crystal structure, as follows.\nArg79 is located within the 78QRATTP83 hexapeptide defined as essential by our deletion analysis; Arg79 forms a salt bridge to Glu150 (Figure 6A), which is located within the essential C-terminal pentapeptide 146WSVEE150. Yet alanine mutation of either Arg79 or Glu150 was benign in vivo (Figure 6B), signifying that this salt bridge is dispensable and that one or more other constituents of the 78QRATTP83 and 146WSVEE150 peptides must be essential for Rpo26 function. In the case of the proximal peptide segment, we suspect that the key contributions are the hydrogen bonds of the main-chain Thr82 and Tyr84 carbonyls to the terminal guanidinium nitrogens of the essential Arg136 side chain (Figure 6A). For the distal 146WSVEE150 peptide, the Trp146 side chain is the likely key constituent, insofar as Trp146 is the focus of an extensive interaction network; it forms a cation–π–cation sandwich between Arg79 and Arg136 (Figure 6A) and it donates a hydrogen bond from Nε to the Glu144-Oε1 atom.\nThe essential Glu89 side chain, located in the first α-helix, forms a bidentate ion pair to the Nε and NH2 atoms of the essential Arg136 side chain, which is situated in the first β-strand (Figure 6A). Glu89 also receives a hydrogen bond to Oε1 from the main-chain amide of Thr86. We surmise that the Glu89-Arg136 salt bridge and the atomic contacts that Glu89 and Arg136 make to the main-chain of the 82TPYMT86 peptide loop preceding the first α-helix are necessary for Rpo26 folding and function. A conservative R136K mutation in RPO26 elicits a temperature-sensitive growth defect (Nouraini et al. 1996a).\nArg135 and Asp145 are situated on the opposite face of the β-hairpin, where they form an interstrand salt bridge (Figure 6A). It was noteworthy that whereas subtracting the Asp145 side chain had no apparent impact on cell growth, the loss of Arg135 was lethal. Thus, the Asp-Arg salt bridge is not essential for Rpo26 activity. Arg135 forms a cation-π stack on Phe143 (Figure 6A), and we suspect that this cation-π interaction accounts for the essentiality of Arg135. Consistent with this idea, replacing Arg135 with lysine, which would, in principle, preserve the cation-π interaction, had no effect on yeast growth (Nouraini et al. 1996a).\nThe essential Glu124 side chain participates in a network of ionic and hydrogen bond contacts involving the two α-helices and the connecting loop (Figure 6A). Glu124 (in α1) makes a bidentate salt bridge to Arg97 (in α1 and conditionally essential at 18°) and accepts a hydrogen bond from the main-chain amide of Phe108 (in the loop). Arg97, in turn, donates hydrogen bonds to Gln100 and the main-chain carbonyl of Pro106. It was shown previously that replacing Gln110 with arginine results in cold-sensitive and temperature-sensitive growth defects (Tan et al. 2003).","divisions":[{"label":"title","span":{"begin":0,"end":89}},{"label":"p","span":{"begin":90,"end":1124}},{"label":"p","span":{"begin":1125,"end":2123}},{"label":"p","span":{"begin":2124,"end":2732}},{"label":"p","span":{"begin":2733,"end":3374}}],"tracks":[{"project":"2_test","denotations":[{"id":"25911228-12697831-43354280","span":{"begin":3937,"end":3941},"obj":"12697831"}],"attributes":[{"subj":"25911228-12697831-43354280","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93dcec","default":true}]}]}}