RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg 2؉ -sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region of asr (acid shock RNA) and another including the promoter for csgD (a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of the asr gene was induced 10-to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg 2؉ led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast, transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade.

Rpb4-Rpb7, a dissociable subcomplex of RNA polymerase II (pol II), is required for transcription initiation. To understand the role of Rpb7 in transcription initiation or other processes in transcription, we carried out a two-hybrid screen for proteins that interact with Rpb7 of the fission yeast Schizosaccharomyces pombe. The screen identified the S.pombe homolog of the Saccharomyces cerevisiae Nrd1, an RNA-binding protein implicated in 3' end formation of small nucleolar and small nuclear RNAs transcribed by pol II. The S.pombe protein, named Seb1 for seven binding, was essential for cell viability, and bound directly to Rpb7 in vitro. Saccharomyces cerevisiae Rpb7 also interacted with Nrd1, indicating that the interaction is conserved in evolution. Glu166 and/or Asp167 of S.pombe Rpb7, residues near the C-terminus of the 172 amino acid protein, were found to be important for its interaction with Seb1. Our results suggest that Rpb7 may function to anchor a processing factor to the pol II apparatus, thereby coupling RNA processing to transcription. The role for Rpb7 is consistent with its location in the pol II complex determined by recent structural studies.

RNA polymerase II (pol II) purified from the fission yeast Schizosaccharomyces pombe was previously reported to be associated with the general transcription factor TFIIF and the C-terminal domain phosphatase Fcp1, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has recently been implicated in transcriptional activation in human cells. Here, we provide evidence that the Rpb7 subunit of pol II interacts with GAPDH. Two-hybrid screen identified GAPDH as an Rpb7-binding protein. In addition, GAPDH was affinity-purified from S. pombe extract by using an Rpb4/Rpb7-coupled column. We also identified actin as a pol II-associated protein and revealed the interaction between actin and Rpb7.

We examined the e#ects of extraction conditions on the anti-allergic components, epigallocatechin---O-(--O-methyl) gallate (EGCG-ῌMe) and strictinin during the manufacture of anti-allergic 'Benifuuki' green tea beverage. Ester-type catechins were extracted at a constant ratio to total catechin content and were easy to epimerize. After a long extraction time at above 3*῍, large amounts of epimer were rapidly produced. Moreover, the ratio of EGCG-ῌMe to polyphenol content was nearly constant, indicating that this ratio could be an essential index during the manufacture of tea beverages. EGCG-ῌMe easily isomerizes at temperatures above 3/῍. From the ratios of EGCG-ῌMe to polyphenol, it was concluded that, at a fixed extraction temperature and ,*-fold dilution (W/V), similar extraction e$ciencies are obtained for extractions from 0 to +/ min heated for 3 min at 3*῍ and 3/῍ showed strong histamine release inhibitory e#ects. Extraction temperature strongly contributed to inhibitory e#ect volume had a limited e#ect. We suspect that extraction at high temperature promoted EGCG-ῌMe extraction e$ciency and epimerization to GCG-ῌMe.

No abstract