|Dr. Dvir's Research: Molecular Mechanisms of Gene Transcription|
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In Dr. Dvir’s lab, we choose a biochemical approach to understand the underlying mechanism of transcription by RNA polymerase II. In principle, we break down cells and reconstitute a transcription reaction from purified cellular components (yes, it works!). There are two basic questions that one can ask:
We are beginning to have some good answers for these questions. RNA polymerase II requires a template DNA that includes a “core promoter”, and an ensemble of five nuclear proteins, called “the general transcription factors”. The illustrations in the box below highlight these molecular components and what is known about the key steps that are part of this process.
In Dr. Dvir’s lab, we study the very early stages of transcription. Following the binding of the polymerase at the promoter, transcription begins with the formation of first phosphodiester bond, which is also called transcription initiation. The formation of the next 15 phosphodiester bonds comprises the next stage on transcription called promoter escape. Apart from RNA polymerase II, the rest of the proteins that are involved in transcription during these two stages are different from the ones that participate in elongation, the latter part of transcription.
Promoter escape is of particular interest to us because there is some evidence that like initiation, it is a regulatory stage in gene expression. Regulated stages in a process are often rate-limiting due to slow steps and usually have specific cofactor requirements. During promoter escape, which has been shown to have both of the aforementioned characteristics of regulatory process stages, the transcription complex undergoes changes in composition which affect the function of the complex. Additionally, the transcription complex has been shown to respond to regulatory mechanisms of specific gene expression.
Early Steps in mRNA Synthesis
|Most Class II promoters contain an eight-nucleotide sequence dubbed the “TATA box”, located approximately 25 bases upstream of the transcription initiation site (-25 position). The TATA box is specifically recognized by the polypeptide transcription factor TFIID, forming tight protein-DNA interactions between its amino acid residues and the DNA bases. The binding of TFIID is the most specific protein-DNA interaction event in promoter recognition. Another area of importance is located around the +1 position (the “Initiator” element), which will be accessed directly by the polymerase.|
|The binding of TFIID at the TATA box allows the association of two addition transcription factors, TFIIB and TFIIF, to form a protein-DNA complex to which the RNA polymerase can bind. The location of the TATA box and the specific assembly of the transcription factors with the polymerase help position its catalytic site directly across +1 for transcription. This pre-initiation complex is dubbed “closed complex” to indicate that strand separation in the DNA surrounding the initiation site has not yet occurred.|
|TFIIH is a transcription factor that can catalyze open complex formation, paving the way to initiation of transcription. Its association with the proteins of the closed pre-initiation complex requires prior binding of another transcription factor, TFIIE. If ATP is present, promoter opening occurs.|
|Three ATP-dependent activities reside in the multi-polypeptide TFIIH: One is a protein kinase, the other two are DNA-helicase activities. Opening the double-stranded structure of the DNA at the initiation site is catalyzed by the DNA-helicase subunits, using ATP hydrolysis to provide energy. The “transcription bubble” forms.|
|Transcription initiation is defined as the formation of a phosphodiester bond between the first two nucleotides of the RNA transcript. To accomplish this step, the transcription bubble needs to be maintained open, and nucleotides complementary to the sequence of the coding strand in the DNA must be available.|
|Following the formation of the first phosphodiester bond in the new RNA, additional nucleotides will be added according to the sequence of the template strand. The polymerase slides forward along the template, and the RNA is growing in length. However, before the RNA has reached a size of 15 nucleotides, the transcription complex is functionally unstable, i.e., transcript elongation is often interrupted and a new transcription cycle has to be established. The occurrence of these unsuccessful initiation attempts decrease dramatically once the RNA has reached a length greater than 15|
|While the polymerase continues to slide forward along the DNA template, elongating the RNA, it is clear that most; if not all of the transcription factors of the initiation complex are no longer associated with it. We are not quite sure when and how this happens. It is also known that during the elongation phase, a different set of transcription factors becomes associated with the polymerase.|
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