Brf is the TFIIB-related component of Saccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB). An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription. The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contributed by a segment of its N-proximal, TFIIB-homologous half. The diverse activities of Brf were also reconstituted from combined fragments. The fragments spanning amino acids 1 to 282 and 284 to 596 were found to assemble into TFIIIB-DNA and TFIIIC-TFIIIB-DNA complexes that were very stable, transcriptionally highly active, and indistinguishable (by in vitro footprinting) from complexes formed with intact Brf. The proximities of the individual halves of split Brf to DNA were extensively mapped by photochemical cross-linking of the TFIIIB-DNA complex. We also identified sites of interaction of Brf fragments with TATA-binding protein (TBP), taking advantage of a recently completed mutational analysis of the TBP surface. The constraints established by these analyses specify a global model of the functional segments of Brf and how they fit into the structure of the TFIIIB-DNA complex.Eukaryotic nuclear and archaeal transcription apparatuses share an essential common feature: the RNA polymerase (pol) is brought to the transcriptional start site by a DNA-bound assembly of transcription initiation proteins. The promotermarking assembly of archaeal RNA polymerases and the core promoter-marking assembly of eukaryotic pol II each consist of two proteins: TATA-binding protein (TBP) and the phylogenetically related transcription factor B (TFB) or TFIIB (for archaeal or pol II transcription systems, respectively) (43).The situation differs only slightly for transcription by pol III, whose promoter-marking assembly, TFIIIB, is composed of three subunits: TBP, Brf (named for its relatedness to TFIIB) (7,11,33), and BЉ (18,24). In Saccharomyces cerevisiae, the three components of TFIIIB are held together by a more stable interaction between Brf and TBP (which together make up the BЈ component of TFIIIB) and by a weaker interaction between TBP-DNA-bound Brf and BЉ (17, 22); a weaker direct BЉ-TBP interaction has also been noted (12, 37). Each of these TFIIIB components is required for all pol III transcription in yeast. Homologs of Brf and BЉ serve as components of the pol III system of humans (42, 44) but are not ubiquitously required. It appears instead that specialized alternative promoter-marking assemblies participate in different classes of human pol III promoters (16,34,42,48).The homology relationship of Brf with TFIIB is confined to the N-terminal half of the much larger Brf; similarities between the C-proximal half of Brf and proteins of the pol II transcription initiation apparatus have not been discerned, at least at the level of...
The yeast RNA polymerase III transcription machinery consists of three transcription initiation factors, TFIIIA, TFIIIB, and TFIIIC (reviewed in references 15, 24, 28, 45, and 47). A fourth component generates quantitatively more efficient transcription in vitro under certain conditions (13, 41), perhaps by facilitating protein refolding or protein-protein association. The functions of TFIIIB are central to this transcription machinery, because TFIIIB directly recruits RNA polymerase III (pol III) to the transcriptional start site and because TFIIIB and RNA pol III alone suffice for transcriptional initiation (26). TFIIIB has three components, TATA-binding protein (TBP), Brf, and BЉ (8,12,22,27,28,36,40,41); TBP and a protein related to yeast Brf are also components of mammalian TFIIIB; it remains to be seen whether a protein analogous to yeast BЉ also exists in higher eukaryotes (11,35,37,42,43). TBP and Brf, together designated BЈ, are readily separable from BЉ during conventional protein purification (25), suggesting that these two components of TFIIIB are somewhat loosely associated (22), although their binding can be detected by immuno-coprecipitation (39) and by affinity chromatography (23).TFIIIB plays a role in transcription by pol III that TFIIB and TFIID (or TFIIB and TBP) together play in pol II transcription. Like TFIIB and TBP (9, 32, 38), TFIIIB occupies upstream sites on pol III genes that specify the locations of transcriptional start sites. TFIIIB is brought to these sites either through the mediation of an assembly factor, TFIIIC, or by autonomous recognition of specific DNA sequence mediated by its TBP.The genes encoding the subunits of yeast TFIIIB have been identified (28,40,41), making the interactions of these components and their roles in initiation of transcription by pol III far more accessible to analysis by biochemical and molecular genetic methods. When Brf is made in Escherichia coli, internal initiation of translation (34) and proteolytic processing generate numerous fragments. Attachment of an affinity tag at the C-terminal end of Brf allows a subset of these products, which constitute a natural N-terminal deletion series, to be purified. The particular interest of such a series lies in the fact that the homology to TFIIB is confined to the N-terminal half of Brf. Two of the N-deletion forms of Brf that are compared in the experiments below progressively remove the N-terminal zinc finger and the first TFIIB-related repeat in the first instance and both TFIIB homologous repeats together with a segment that is somewhat conserved among fungal Brf (Brf homology I) in the second case. A third deletion variant lacks all of the above and 35 additional amino acids.We have compared these deletion proteins with intact Brf for the ability to interact with the other components of TFIIIB, for the ability to participate in effective recruitment of pol III to a U6 gene promoter, and for ability to be recruited to a tRNA gene by TFIIIC. Our findings suggest a functional complementarity between Br...
The Cuatro Cié negas Basin (CCB) in the central part of the Chihuahan desert (Coahuila, Mexico) hosts a wide diversity of microorganisms contained within springs thought to be geomorphological relics of an ancient sea. A major question remaining to be answered is whether bacteria from CCB are ancient marine bacteria that adapted to an oligotrophic system poor in NaCl, rich in sulfates, and with extremely low phosphorus levels (<0.3 M). Here, we report the complete genome sequence of Bacillus coahuilensis, a sporulating bacterium isolated from the water column of a desiccation lagoon in CCB. At 3.35 Megabases this is the smallest genome sequenced to date of a Bacillus species and provides insights into the origin, evolution, and adaptation of B. coahuilensis to the CCB environment. We propose that the size and complexity of the B. coahuilensis genome reflects the adaptation of an ancient marine bacterium to a novel environment, providing support to a ''marine isolation origin hypothesis'' that is consistent with the geology of CCB. This genomic adaptation includes the acquisition through horizontal gene transfer of genes involved in phosphorous utilization efficiency and adaptation to high-light environments. The B. coahuilensis genome sequence also revealed important ecological features of the bacterial community in CCB and offers opportunities for a unique glimpse of a microbe-dominated world last seen in the Precambrian.evolution ͉ genomic adaptation ͉ horizontal gene transfer ͉ phosphorus stress ͉ sulfolipids T he Cuatro Ciénegas Basin (CCB) is located in a valley Ϸ740 m above sea level in the state of Coahuila, Mexico, that measures Ϸ30 km by 40 km and is surrounded by high mountains (Ͼ3,000 m) (Fig. 1). CCB is an enclosed evaporitic basin that receives Ϸ150 mm of annual precipitation. Despite the dry climate of the valley, the CCB harbors an extensive system of springs, streams, and pools (1). The CCB ecosystem is not only characterized by a high endemism of macrooganisms and biodiversity of microorganisms (1, 2), but also by extremely oligotrophic waters that are unable to sustain algal growth, making microbial mats the base of the food web (3). In particular, phosphorus (P) levels in CCB appear to be rather low, because they were below the level of detection of several methods used (0.3 M) and the extremely high biomass C:P and N:P ratios (Ͼ100 by moles) previously reported for CCB stromatolites (3, 4). Unlike the present sea, the Churince spring water is poor in NaCl and carbonates, but it is rich in sulfates, magnesium, and calcium (4). Characterization of the microbiological diversity by sequencing 16S rRNA genes revealed that nearly half of the phylotypes from the CCB were closely related to bacteria from marine environments (2). Bacillus coahuilensis is a free-living, spore-forming bacteria isolated from the water column of a shallow desiccation lagoon in the Churince system at CCB (4) (Fig. 1 A and B). A molecular phylogenetic analysis of 16S rRNA sequences indicates that B. coahuilensis is closely ...
A moderately halophilic, Gram-positive and rod-shaped bacterium, strain m4-4 T , was isolated from a Chihuahuan desert lagoon in Cuatro Cié negas, Coahuila, Mexico. Strain m4-4T was found to grow optimally at 30-37 6C, pH 7.0-8.0 and 5 % NaCl and to tolerate from 0.5 % to 10 % NaCl. It was shown to be aerobic. The genomic DNA G+C content was about 37 mol%. Strain m4-4 T exhibited minimal or no growth on most sugars tested. Its major cellular fatty acids were C 14 : 0 , C 16 : 0 and C 18 : 1 . Based on phylogenetic analysis of 16S rRNA and recA gene sequences, we observed that the closest relatives of the isolate are moderately halophilic Bacillus species, with 16S rRNA gene sequence similarity ranging from 96.6 to 97.4 % (Bacillus marisflavi, Bacillus aquimaris and Bacillus vietnamensis). Additionally, using genomic data it was determined that the type strain contains a total of nine rRNA operons with three slightly different sequences. On the basis of phenotypic and molecular properties, strain m4-4 T represents a novel species within the genus Bacillus, for which the name Bacillus coahuilensis sp. nov. is proposed, with the type strain m4-4 T (5NRRL B-41737 T 5CECT 7197 T ).
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