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The predominant organizational theme by which the transcription machinery and chromatin regulators are positioned within promoter regions or throughout genes in a genome is largely unknown. We mapped the genomic location of diverse representative components of the gene regulatory machinery in Saccharomyces cerevisiae to an experimental resolution of <40 bp. Sequence-specific gene regulators, chromatin regulators, mediator, and RNA polymerase (Pol) II were found primarily near the downstream border from the "-1" nucleosome, which abuts against the approximately 140-bp nucleosome-free promoter region (NFR). General transcription factors TFIIA, -B, -D, -E, -F, -H were located near the downstream edge from the NFR. The -1 nucleosome dissociated upon Pol II recruitment, but not upon recruitment of only TBP and TFIIB. The position of many sequence-specific regulators in promoter regions correlated with the position of specific remodeling complexes, potentially reflecting functional interactions. Taken together the findings suggest that the combined action of activators and chromatin remodeling complexes remove the -1 nucleosome after the preinitiation complex (PIC) has partially assembled, but before or concomitant with Pol II recruitment. We find PIC assembly, which includes Pol II recruitment, to be a significant rate-limiting step during transcription, but that additional gene-specific rate-limiting steps associated with Pol II occur after recruitment.
The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIB(ZR)) and a carboxy-terminal core (TFIIB(CORE)). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIB(ZR) that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIB(ZR) surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIB(ZR) domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters-pol II recruitment-has changed in sequence specificity during eukaryotic evolution.
Interaction between the TATA box-binding protein TBP and TFIIB is critical for transcription in vitro. An altered-specificity TBP-TFIIB interaction was rationally designed and linked in sequence to an altered-specificity TATA box-TBP interaction to study how TBP and TFIIB function together to support transcription in human cells. The activity of this altered-specificity TATA-TBP-TFIIB array demonstrated that many activators use the known TBP-TFIIB interaction to stimulate transcription. One activator, however, derived from a glutamine-rich activation domain of Sp1, activated transcription independently of this interaction. These results reveal that selectivity in activator function in vivo can be achieved through differential use of TBP and TFIIB.