Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites. In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites. This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in the in vitro systems. Therefore, Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions. Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid. 6-Azauracil also induced IMD2 gene expression in both these mutant strains and the wild type. Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37°C, a restrictive temperature for a strain bearing double null mutations of spt4 and S-II. In contrast, strains bearing S-II mutations defective in both cleavage and readthrough stimulation had phenotypes similar to those of an S-II null mutant. However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain. These results suggest that cleavage, but not readthrough, stimulation activity is responsible for all three biologic functions of S-II (i.e. suppression of 6-azauracil sensitivity, induction of the IMD2 gene, and suppression of temperature sensitivity of spt4 null mutant).Transcription elongation factor S-II, originally purified from mouse Ehrlich ascites tumor cells, is an RNA polymerase IIstimulating factor in promoter-independent RNA synthesis (1). It has been found in all eukaryotes thus far investigated, and its primary structure is highly conserved (2-7). S-II is a unique transcription elongation factor that promotes transcript elongation through transcription arrest sites found in genes (8 -11). The molecular mechanism of this phenomenon has been investigated extensively in in vitro systems, and it has been shown that S-II stimulates the nuclease activity of RNA polymerase II, which then cleaves the nascent transcript. Then the 3Ј-end of the nascent RNA is realigned with the catalytic site of RNA polymerase II, and the transcription elongation complex tries reading through the arrest site again (11-14). The cleavage stimulation activity of S-II can be separated from its readthrough stimulating activity; although the cleavage stimulation activity is essential, it is not sufficient to promote readthrough of RNA polymerase II in vitro (15).There have been several reports describing the function of S-II in Saccharomyces cerevisiae. In several yeast strains bear null mutations of the genes encoding the transcription elongation machinery, such as spt4⌬, r...
A similar approach was taken for mutations in the  subunit key region; consistent with its bulk phase ATPase activities, F 1 with the Ser-174 to Phe substitution (S174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120°positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was ϳ30-fold higher than the K m value for ATP. A Gly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of S174F. The revertant (G149A/S174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant G149A/S174L. These results indicate that the domain between -sheet 4 (Ser-174) and P-loop (Gly-149) is important to drive rotation.A ubiquitous ATP synthase (FoF 1 ) synthesizes ATP coupled with an electrochemical proton gradient formed by a respiratory chain (for reviews, see Refs 1-5). FoF 1 consists of a catalytic sector, F 1 (␣ 3  3 ␥␦⑀), and a membrane-embedded proton pathway, Fo (ab 2 c 10 ), and can reversibly transport protons coupled with ATP hydrolysis. The ␣ and  subunits form a catalytic hexamer (␣ 3  3 ), the central space of which is occupied by the ␥ subunit ␣-helices. ATP is synthesized or hydrolyzed cooperatively at a catalytic site in each  subunit as the binding change mechanism predicts (2). The ␥ subunit rotation in ␣ 3  3 has been supported by biochemical studies (1, 6, 7), a crystal structure of the ␣ 3  3 ␥ complex (8), and video recorded using an actin filament as a probe (9, 10). Consistent with ATP-dependent proton translocation, a ␥⑀c 10 complex rotated relative to the ␣ 3  3 ␦ab 2 in the purified FoF 1 (11-14) or its membrane-bound form (15, 16). The rotation of FoF 1 in liposomes has been revealed by means of single molecule fluorescence resonance energy transfer (17).Counterclockwise rotation of the ␥ subunit has been studied more recently with probes giving low viscous drag such as colloidal gold (14,18,19). The three 120°steps in one revolution of Bacillus F 1 were first observed using an actin filament (20), and later the single 120°step was further subdivided into two substeps (80°and 40°) using gold beads that allow finer observation and analysis (14,19,21). The substeps with larger displacement angles (80°) and smaller substeps (40°) are assigned to ATP binding and hydrolysis/product release steps, respectively (19,21,22). We have observed that the rotation speed of beads attached to the Escherichia coli ␥ subunit varied, reflecting stochastic fluctuations (18,23). Although the average speeds were dependent on the diameter of beads, 40-and 60-nm diameter beads rotated with essentially the same rate (18), suggesting that their rotation speeds were close to that of...
In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.
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