Mrna Synthesis

Coordination of gene expression determines the cellular transcriptome, which in turn specifies the nature of the proteomes and defines the cellular function. In metazoan organisms formation of a preinitiation complex at the right time and at the right promoter is a prerequisite to executing the correct programs of mRNA synthesis. This involves the interplay of many gene specific transcription factors and an array of cis-regulatory DNA elements located in the promoter enhancer regions of various tissue and developmental stage restricted genes.

(1) Eukaryotic Promoter: A Multi-Component Structure

Control of gene transcription is one of the major regulatory mechanisms operative in a particular cell type. Average eukaryotic cells contain between 5000 to 35,000 genes distributed along their chromosome. Three different DNA-dependent RNA polymerases are required for eukaryotic transcription, of which:

- RNA polymerase I transcribes ribosomal RNA in the nucleolus and is responsible for approximately 50-70% of the total RNA polymerase activity.

- RNA polymerase II synthesizes messenger RNA in the nucleoplasm and accounts for approximately 20-40% of total RNA polymerase activity.

- RNA polymerase III synthesizes tRNA, 5SRNA and U6snRNA in the nucleoplasm and accounts for 10% of the total RNA polymerase activity.

1.1.1: Core promoter elements define the site for the assembly of the transcription preinitiation complex (PIC) and include a TATA sequence, located upstream of the transcription start site and an initiator sequence (Inr), encompassing the start site. Promoters can include either a TATA box or an Inr sequence or both. A third core element, the downstream promoter element (DPE), was initially described in Drosophila and is located about 30 bp downstream of the start site. The DPE appears to function, in conjunction with the Inr element, as a TFIID binding site in TATA-less promoters.

TATA Elements: In higher eukaryotes, the TATA element is located at a distance of 25 to 30 bp from the start site while in S. cerevisiae they are typically located 40 to 120 bp upstream of the transcription initiation site. The TATA sequence is the binding site for the TATA binding protein (TBP). TBP-TATA association nucleates the assembly of an approximately 4-MDa transcription preinitiation complex- a step that can be rate limiting for transcription initiation in vivo.

Initiator element: Initiator elements (Inr) are DNA sequences encompassing transcription start sites. Experiments to determine the relationship between TATA and the Inr established that the TATA element defines the window within which initiation can occur but that specific sequences within the window define the Inr element.

1.1.2: Regulatory elements: These are gene-specific sequences that are located upstream of the core promoter and control the rate of transcription initiation. They include both enhancer/upstream activation sequences (UAS) and repressor/upstream repression sequences (URS).

Enhancers/UAS: Activator sequences in yeast are known as UAS whereas its counterpart in metazoan cells is known as enhancer. These DNA sequences function as binding sites for specific transcriptional activators. Enhancers can function in either orientation and at variable distances from the core promoter. Once associated with their cognate UAS elements, transcriptional activators facilitate assembly of the PIC, either by direct contact with the general transcription factors (GTFs) or indirectly through coactivators, which in some cases mediate activator-GTF interactions.

Repressors/URS: These are binding sites for gene-specific transcriptional repressors. Repressor/URS complexes can impair transcription by several different mechanisms including, interference with activator-UAS binding; interference with the activation domain of an activator-UAS complex; or by contact with the core transcriptional machinery, a process analogous to activation, albeit with opposite effects. Repressor complexes can also mediate repression indirectly by recruiting another complex that targets either the core transcriptional machinery or histones. Transcriptional repression associated with histone deacetylation is example of this type of repression.

Poly (dA-dT) Elements: Homopolymeric dA-dT sequences are a common feature of yeast promoters and in several cases have been shown to be required for normal levels of transcription in vivo. Poly (dA-dT) sequences have distinct structural characteristics that impair nucleosome assembly or stability, which led to the proposal that poly (dA-dT) sequences function as promoter elements based on their intrinsic structure, rather than as conventional UAS elements to which sequence-specific transcription factors bind.

1.2: RNA Polymerase II: RNA polymerase II is a multisubunit structure, which is composed of 12 subunits encoded by the RPB1 to RPB12 genes. There is extensive structural conservation among the subunits of eukaryotic RNA Pol II. The two largest RNA Pol II subunits, Rpb1 (200 kDa) and Rpb2 (150 kDa) are the most highly conserved subunits. Rpb3 is related to the a subunit of bacterial RNA polymerase and is involved in RNA Pol II assembly. The Rpb1, Rpb2, Rpb3, and Rpb11 subunits of RNA Pol II are homologous to subunits of RNA polymerases I and III. Moreover, five subunits- Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are common to all three RNA polymerases. Only Rpb4, Rpb7, and Rpb9 are unique to RNA Polymerase II. Thus RNA polymerases are assembled from common as well as class-specific subunits. Carboxy-terminal repeats domain: A unique feature of the largest RNA Pol II subunit is the presence of tandem repeats of a heptapeptide sequence at its carboxy-terminus. This carboxy-terminal repeats domain (CTD) has the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser that is highly conserved among eukaryotic organisms. Although the CTD is a ubiquitous feature of RNA Pol II, the repeat length varies. For example, yeast Rpb1 includes 26 or 27 repeats, C. elegans CTD has 34 repeats, Drosophila CTD has 43 repeats, and human CTD 52 repeats, suggesting that repeat length increases with increasing genome complexity.

1.3: Transcription Regulation of Eukaryotic Protein-Coding Genes is An Orchestrated Process: Transcription regulation in eukaryotes is a coordinated process and requires the concerted functions of multiple proteins or transcription factors. These factors can be classified into three groups:

General/basal transcription factors which are ubiquitous and include RNA polymerase II (Pol II) and a set of accessory general transcription initiation factors (GTFs) that bind to core promoter DNA elements (e.g., TATA box, initiator) and allow the specific recruitment of Pol II to the core promoter of all class II genes. GTFs functions are conserved among eukaryotic organisms.

Sequence-specific DNA-binding transcription regulators (i.e., activators and Repressors) which bind to proximal promoter elements and/or distal regulatory sequences (i.e., enhancers and silencers) and modulate the rate of transcription of specific genes in a tissue and developmental stage-specific manner or in response to physiological or environmental stimuli.

Cofactors/coregulators (coactivators and corepressors), which interact with general or gene specific transcriptional regulators and play essential roles in mediating or facilitating their effects on transcriptional machinery. They act either via direct physical interactions with GTFs and Pol II or indirectly through modification of chromatin structure.

1.4: Eukaryotic Transcription Involves Multi-Protein-RNA Polymerase II Complexes: The GTFs include TBP, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH and were identified biochemically as the factors required for accurate in vitro transcription initiation of adenoviral promoters by RNA Pol II. Order-of-addition experiments demonstrated that the assembly of preinitiation complex is nucleated by the TBP binding to the TATA element followed by binding of TFIIA, TFIIB, RNA Pol II-TFIIF, TFIIE, and TFIIH. Amongst all these general transcription factors, TBP is a universal one, required for the initiation by all three eukaryotic RNA polymerases.

TATA binding protein (TBP) is a universal protein: TBP is a subunit of TFIID, multisubunit complex composed of TBP and TBP-associated factors. TBP is an essential transcription factor that affects promoter recognition and is required for the expression of many, if not all, genes in vivo. While TBP functions at the level of basal transcription in vitro, in metazoan cells, TFIID is required for mediating response to transcriptional activators. TBP is an essential subunit of the RNA Pol I transcription factor SL1. TBP was also identified as a subunit of TFIIIB, a component of RNA Pol III machinery. Deduced TBP amino acid sequence revealed two direct repeats encompassing the C-terminal two-thirds of the protein. Subsequent comparison with the phylogenetic series of TBP sequences demonstrated that the C-terminal direct repeats are highly conserved among vertebrates. Unlike other DNA binding proteins, TBP recognizes its binding site through minor groove contacts. The crystal structure of Arabidopsis TBP revealed a remarkable structure containing a new DNA binding fold, resembling a molecular saddle that sits on the DNA. TBP plays a critical role in the transcriptional activation by direct contact between TBP and the activation domains of many gene-specific activators. Complex formation between TBP and the TATA element occurs by a two-step mechanism involving a slow TBP-TATA association followed by a rapid conformational change. Alternatively, the rate-limiting step in TBP-TATA complex formation is the dissociation of TBP-TBP dimers. TFIID also dimerizes in the absence of DNA, with dimer formation mediated by TBP-TBP association. TBP has paralogue genes (TRF1, TRF2, TRF3,) and these TBP-like proteins are often expressed in a cell type- or tissue-specific pattern.

All these proteins interact with a multitude of coregulatory partners to elicit gene-specific responses and drive distinct biological processes.

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