PMC:4502370 / 29007-34724 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"N- and C-terminal truncations of Rpo26 delineate a minimal functional domain\nThe crystal structure of yeast RNA polymerase II (Cramer et al. 2001) revealed the fold of the C-terminal segment of Rpo26 from amino acids 72 to 155, which comprises two α-helices and a β-hairpin (Figure 5). The N-terminal 71-amino-acid segment was disordered in the Pol II structure. In the recent crystal structure of yeast Pol I, the N-terminal 54-amino-acid segment of Rpo26 was disordered and the segment from amino acids 55 to 71 comprised an α-helix (Fernández-Tornero et al. 2013). A previous study had shown that deleting 42 amino acids from the N-terminus of Rpo26 did not affect the viability of yeast cells when the truncated RPO26-∆42 allele was driven by the strong GAL10 promoter in galactose-containing medium; however, deletion of 84 amino acids from the Rpo26 N-terminus was lethal (Nouraini et al. 1996a).\nFigure 5 N- and C-terminal truncations of Rpo26 delineate a minimal functional domain for tgs1∆ suppression. (Left) Tertiary structure of Rpo26-(72-155), from the yeast Pol II crystal structure (pdb 1I3Q), with the N and C termini indicated. (Bottom right) The amino acid sequence of yeast Rpo26. The C-terminal segment visualized in the Pol II crystal structure is in black font; the disordered N-terminal segment is in red font. The margins of the N- and C-terminal truncations are denoted by forward and reverse arrows. For the N-terminal deletions, the arrows specify the residues that were mutated to methionine to initiate the truncated proteins. Black arrows denote the truncations that allow the mutants to restore tgs1Δ growth at 18°, whereas the gray arrows denote the truncations that disable tgs1∆ suppression. (Top right) tgs1Δ cells were transformed with a CEN LEU2 plasmid bearing wild-type TGS1 (positive control), an empty 2-μ LEU2 TPI1 vector (negative control), or 2-μ LEU2 TPI1-RPO26 plasmids bearing wild-type RPO26 or the indicated truncation mutants. Leu+ transformants were selected at 30° and then tested for growth at 18° by spotting serial 10-fold dilutions of liquid cultures (grown at 30° in SD-Leu medium) on SD-Leu agar plates. The plates were photographed after incubation for 7 d at 18°. Here, we tested the effects of finer incremental N- and C-terminal truncations on the in vivo activity of Rpo26, using two genetic readouts of function: (i) dosage suppression of tgs1∆ and (ii) complementation of rpo26∆. The truncated RPO26 alleles were placed on 2-µ plasmids under the control of the yeast TPI1 promoter. The N-terminal deletion alleles RPO26-(40-155), RPO26-(70-155), and RPO26-(78-155) were as effective as RPO26 in supporting tgs1∆ growth at 18°, whereas RPO26-(1-80), a truncated version encoding just the disordered N-terminal segment of Rpo26, had no salutary effect (Figure 5). The RPO26-(78-155) allele complemented rpo26∆ in a plasmid shuffle assay. RPO26-(78-155) cells grew as well as wild-type RPO26 yeast at 18°, 25°, 30°, and 37°, as gauged by colony size (Figure 6B). We conclude that the N-terminal 77 amino acids are dispensable for Rpo26 function as a subunit of the three nuclear RNA polymerases and as a suppressor of tgs1∆. By contrast, RPO26-(84-155) was a feeble suppressor of tgs1∆ at 18° (Figure 5) and was unable to complement rpo26∆ in the plasmid shuffle assay (not shown), signifying that the 78QRATTP83 peptide is important for Rpo26 activity.\nFigure 6 Structure-guided alanine scan of Rpo26. (A) Annotated structure of Rpo26 (from pdb 1I3Q) highlighting atomic interactions (dashed lines) of selected side chains and main chain atoms (depicted as stick models with beige carbons). Eight residues were targeted for alanine scanning: Arg79, Glu89, Arg97, Glu124, Arg135, Arg136, Asp145, and Glu150. The lethal RPO26-Ala alleles are indicated on the right. (B) rpo26∆ complementation. Yeast strain rpo26Δ p(URA3 CEN RPO26) was transformed with 2-μ LEU2 TPI1-RPO26 plasmids bearing wild-type RPO26, RPO26-(78-155), or the indicated RPO26-Ala mutants. Leu+ transformants were selected at 30° on agar medium containing 5-FOA (1.0 mg/ml) and aliquots of serial 10-fold dilutions of the strains with the specified genotypes were spotted on YPD agar medium. The plates were photographed after incubation for 2 d at 37°, 3 d at 30°, 5 d at 25°, or 7 d at 18°. (C) tgs1∆ suppression. tgs1Δ cells were transformed with a CEN LEU2 plasmid bearing wild-type TGS1 (positive control), an empty 2-μ LEU2 TPI1 vector (negative control), or 2-μ LEU2 TPI1-RPO26 plasmids bearing wild-type RPO26 or the indicated RPO26-Ala mutants. Leu+ transformants were selected at 30° and then tested for growth at 18° by spotting serial 10-fold dilutions of liquid cultures (grown at 30° in SD-Leu medium) on SD-Leu agar plates. The plates were photographed after incubation for 7 d at 18°. We then tested the effects of deleting 5 amino acids or 16 amino acids from the C-terminus of the biologically active Rpo26-(70-155) polypeptide. Whereas RPO26-(70-150) was able to support growth of tgs1∆ cells at 18°, the RPO26-(70-140) allele was not (Figure 5). RPO26-(70-140) failed to complement rpo26∆ (not shown). By contrast, RPO26-(70-150) did complement rpo26∆, albeit with a conditional phenotype whereby RPO26-(70-150) cells grew well at 18° and 25°, formed small colonies at 30°, and failed to grow at 37° (Figure 6B). Thus, the decapeptide segment 141GSFEDWSVEE150 is necessary for Rpo21 function at warmer temperatures. Because a previous study had shown that a nonsense mutant allele encoding Rpo26-(1-145) was unable to complement rpo26∆ (Nouraini et al. 1996a), we can surmise that the pentapeptide 146WSVEE150 contains features essential for Rpo26 activity in vivo.","divisions":[{"label":"title","span":{"begin":0,"end":76}},{"label":"p","span":{"begin":77,"end":902}},{"label":"figure","span":{"begin":903,"end":2224}},{"label":"label","span":{"begin":903,"end":911}},{"label":"caption","span":{"begin":913,"end":2224}},{"label":"p","span":{"begin":913,"end":2224}},{"label":"p","span":{"begin":2225,"end":3416}},{"label":"figure","span":{"begin":3417,"end":4832}},{"label":"label","span":{"begin":3417,"end":3425}},{"label":"caption","span":{"begin":3427,"end":4832}},{"label":"p","span":{"begin":3427,"end":4832}}],"tracks":[{"project":"2_test","denotations":[{"id":"25911228-11313498-43354274","span":{"begin":141,"end":145},"obj":"11313498"},{"id":"25911228-24153184-43354275","span":{"begin":561,"end":565},"obj":"24153184"}],"attributes":[{"subj":"25911228-11313498-43354274","pred":"source","obj":"2_test"},{"subj":"25911228-24153184-43354275","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ecb2","default":true}]}]}}