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With particular emphasis on the biochemistry, discuss the machinery involved in the transcriptional process.
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This solution is an essay detailing the conversion of DNA to RNA. This solution includes all the necessary peer-reviewed citations in the text with full references. This is a 5000 word solution and includes two figures to illustrate the process.
The mechanism of transcription: regulation of the conversion of DNA into RNA
A central dogma of biology is that DNA is transcribed into single-stranded RNA, which is then translated into proteins. Translation of proteins is dependent on fine control of genes. Through the action of various factors that influence the transcription of a gene, a particular protein is ultimately translated. This fine control allows external factors such as changes in the environment or other stresses to dictate what proteins a cell produces.
The genome of every cell codes for every possible protein needed in any cell type in the entire organism but most cells need only a subset of these proteins to conduct the specific functions of an individual cell type. Hence, a cell will only translate proteins only as they are necessary and in response to different cues. There must be some sort of control that allows transcription of genes specific to an environmental stress. There must also be a means of controlling the rate of transcription of a particular gene in a timely manner.
This essay reviews the machinery and the various control mechanisms involved in transcription that are employed by the cell. The process of transcription is a complex mutlistep process that has various checkpoints of control that are examined in this essay. As well, this essay also investigates a few techniques that are used in the study of transcription and gene regulation via transcriptional control.
During transcription a single-stranded piece of RNA is made from a double-stranded piece of DNA. In eukaryotes a gene usually contains coding sequences or exons, interspersed with non-coding sequences or introns. After transcription, the non-coding sequences are removed to form message RNA (mRNA), which is then transferred to the cytoplasm for translation into proteins. A gene also contains specific sequences before and after the transcribed region that are involved in the regulation of transcription. Introns and exons along with these regulatory sequences make up the structure of a gene (Fig. 1). The transcription process involves numerous proteins that act in a concerted fashion to allow RNA polymerase to work (see Shilatifard, 1998). These proteins are all subject to regulation and are points of control in the transcription of a gene.
Three different RNA polymerases (pol I, II, III) synthesize different types of RNA from a DNA template. Pol I, II, and III transcribe ribosomal RNA (rRNA), message RNA (mRNA), and transfer RNA (tRNA), respectively. This review will focus mostly on Pol II and its involvement in the transcription of DNA into mRNA. The Pol II basal machinery binds to a region called the promotor, which is upstream of the DNA transcriptional start site. The promotor region is an important sequence as the strength of binding of the basal machinery to this region determines how frequently a gene will be transcribed. The frequency of gene transcription, as will be discussed further, is dependent on transcription factors and their control. The transcriptional start site is typically designated as +1 and sequences upstream of the transcriptional start site are given a negative designation. The promotor region is found upstream of the transcriptional start site and includes the TATA box, a highly conserved septamer (5'-TATAAAA-3') that is found at about position -25 (Workman and Roeder, 1987).
Transcription can be broken down into three stages: pre-initiation and initiation, followed by elongation, and then termination. These three steps are outlined below and summarized in Figure 2. Pre-initiation and initiation steps happen close together, do not have clear boundaries and will be discussed here as a single stage. Elongation is the actual synthesis of the mRNA strand by the addition of the ribonucleotides adenine, guanine, cytosine and uracil. During synthesis of RNA, instead of adding thymine, uracil residues are added instead. Termination involves the release of Pol II from the DNA template allowing it to make another RNA transcript.
Pre-Initiation and Initiation
The pre-initiation and initiation steps involve protein interactions that unwind the chromatin DNA and facilitate the interaction of the exposed DNA template with RNA polymerase (Ghosh and Van Duyne, 1996). The pre-initiation step involves the formation of the basal machinery from several general transcription factors that allow the binding of Pol II and the initiation of transcription.
One of the first factors to bind does so at the TATA box. The TATA box binding protein (TBP) recognises the septamer and binds at the minor groove of the DNA double helix. TBP looks like a saddle with its inner surface binding to the DNA leaving the outer surface free to interact with other proteins. In fact, eleven other proteins interact, with TBP. TBP together with eight other TBP associated factors (TAFs) forms a large complex known as TFIID (Transcription factor D for pol II) (Ghosh and Van Duyne, 1996). TBP and TFIID are used interchangeably throughout the literature but will be referred to as TBP herein.
The second factor to bind is TFIIA (transcription Factor A for pol II), which binds directly to TBP and aids in stabilizing the interaction between TBP and the DNA (Maldonado et al., 1990). Nogales (2000) suggests from structural observations that TFIIA increases the surface area of the basal complex that interacts with the DNA. TFIIA also functions as an anti-repressor, blocking proteins that de-stabilize the TBP-DNA interaction (Coleman et al., 1999). TBP can form a dimer through its DNA binding domain and thus prevent any unwanted gene transcription. TFIIA can block the dimerization of TBP by promoting its disassociation leaving TBP in monomer form (Coleman et al., 1999). As a result, TFIIA facilitates TBP binding to the DNA promotor and transcription of the gene.
After TFIIA binding, a third factor called TFIIB binds to the growing basal complex. TFIIB binds to TBP directly and aids in stabilizing the binding to the TATA box. Both TFIIB and TFIIA ...
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