Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6A⌬ nhp6B⌬ double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6A⌬ nhp6B⌬ cells at 37°C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6A⌬ nhp6B⌬ cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxilliary factors are required for the optimal transcription of at least some specific Pol III genes.Transcription of small genes by RNA polymerase III (Pol III) in yeast involves a multistep assembly of transcription factors into a preinitiation complex which recruits RNA Pol III (for a review, see reference 35). The A and B blocks found in most Pol III promoters are first recognized by a multisubunit complex called Pol III transcription factor C (TFIIIC). TFIIIC, one of the largest and most complex transcription factors known, has a molecular mass of about 600 kDa and is composed of six subunits. It acts as an assembly factor to direct the binding of the initiation factor TFIIIB to an upstream gene position. Once assembled into a highly stable protein-DNA complex at Pol III promoters, TFIIIB can direct multiple rounds of transcription by Pol III in vitro in the absence of TFIIIC (17,18). TFIIIB is composed of three loosely associated polypeptides, the TATA-binding protein (19), a general transcription factor used by all eukaryotic and archeal RNA polymerases (14, 27); BЉ or TFIIIB90, which displays little homology to other known proteins except for a putative SANT domain (1,20,28,29); and Brf1 or TFIIIB70, which shows 44% similarity to TFIIB in its N-terminal 320 residues (3,7,21).In addition to these basal factors, there are hints that additional components exist which influence transcription efficiency or accuracy. A protein called TFIIIE, which has yet to be characterized, is able to stimulate transcription under certain conditions (9, 29). TFIIIE has been suggested to act by facilitating TFIIIB recruitment, by inducing conformational rearrangements of TFIIIB, or by stabilizing transcription complexes. A partially purified BЉ fraction was found to direct a more efficient and more accurate transcription initiation than the recombinant TFIIIB90 protein (6, 29), but the factors p...
BackgroundThe variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA2 neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA2 composition of the snakes captured in the same areas.Methodology/Principal FindingsWe conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA2s. We used SELDI technology to study the diversity of PLA2 in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA2s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA2 venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains.Conclusions/SignificanceThe association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA2 genome and transcriptome data.
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