Eukaryotic Transcription

In spite of this similarity, however, the details are very different in eukaryotic cells and are summarized as follows. The fundamental process is identical in prokaryotes and eukaryotes, in that an RNA polymerase complex binds to the promoter and initiates transcription at a start site downstream to the promoter. De novo initiation of anRNAchain occurs with a purine nucleotide and creation of a transcription bubble with the open complex. The transcription complex can slide back along the nascent chain and endonucleolytically cleave off the 3´ segment, then moves forward along the DNA template chain; termination occurs at specific regions in the genes.

1. Eukaryotic RNA polymerases contain many more subunits, located in the different regions of the nucleus. Pol I, specific for synthesizing rRNA, is located in the nucleolus, a specialized structure within the nucleus, while Pol II and Pol III are in the nucleoplasm. These enzymes have 8–14 subunits with a total molecular mass >500 kD. The large subunits have some sequence similarity with the bacterial RNA polymerases. RNA polymerases of mitochondria and chloroplasts are phylogenetically closer to bacterial RNA polymerase, commensurate with the fact that the target genes of these enzymes are fewer and much smaller in organelles, which are thought to have arisen by symbiotic acquisition of bacteria by primitive eucaryotes.
   
   
2. The promoter composition and organization of eukaryotic polymerases are quite specific for each polymerase. The promoters of rRNA genes contain a core and an upstream control element which is needed for high promoter activity. Two ancillary factors, UBFl and SLl, bind to these sequences. Although SLl binds only after UBFl in a cooperative fashion, SL1 is a σ-factor with four proteins among which TBP is also required for initiation by the other polymerases. Pol I is akin to Pol III in that it utilizes both upstream and downstream promoters. There are two types of internal promoters with distinct sequence boxes. One transcription factor (TFIII B) is required for initiation of RNA synthesis by Pol III. Other factors (TFIII A and TFIII C) help TFIII B bind to the right location and act as positioning factors for correct localization of Pol III initiation.
   
   Pol II is the most versatile and widely utilized RNA polymerase in vivo and absolutely needs auxiliary, transcription factors (TFII) whose requirement is dependent on the nature of promoters.
   
   
3. The nature of eukaryotic promoters is quite different from the prokaryotic promoters. In addition to the bipartite promoter of Pol I, both Pol II and Pol III have a “TATA box” located about 25 bp upstream of the start site in Pol II responsive genes. The 8-bp sequence consists of only AT base pairs and is surrounded by GC pair-rich sequences. Interestingly, the TATA box is quite similar to the −10 sequence in E. coli promoters.
   
   There is a second element called a CAAT box, usually about −15 bp 5´ of the TATA box. Alternatively a GC rich sequence is present in some promoters, often at position −90. The consensus GC box sequence is GGGCGG, of which multiple copies are often present and occur in both orientations. These elements are not all present in all promoters; it appears that they work in a “mix and match” fashion. These boxes, and also a octamer box, bind to specific trans-acting factors and are engaged in multiple protein interactions among themselves as well as with components of the RNA Pol II holoenzyme.
   
   There is no significant homology among transcription start sites of various genes, except for the tendency for the first base in the transcript to be an A flanked on either side by pyrimidines. This region is defined as the initiator.
   
   The first step in transcriptional initiation of a TATA containing promoter is the binding of the factor TFIID to the region upstream of the TATA site. The TATA-binding protein, TBP, which specifically binds to the TATA box, is a component of the TFIID complex, along with other proteins collectively called TAFs (TBP-associated factors). TAFs can be variable in the TFIID complex, both in species and amounts, and provide the promoter specificity for initiation. Some TAFs are tissue specific. TFIID has a molecular mass of 800 kD, containing 1 TBP and 11 TAFs. TBP acts as a positioning factor and is able to interact with a wide variety of proteins, including Pol II and Pol III. It binds to the minor groove of the DNA double helix and makes contact with other factors which mostly bind to the major groove and can make multiple contacts. By bending the DNA at the binding site, it appears to bring the factors and RNA polymerase into closer proximity.
   
   Although TBP is utilized by both Pol II and Pol III, TFIID is the specific complex for Pol II recognition of a promoter. Other transcription factors (e.g., TFIIA) bind to the TFIID promoter complex and cover increasing segments of DNA. In addition to TFIIA, these include TFIIE, TFIIF, TFIIH, and TFIIJ. Most of the TFII factors are released from the transcription complex before Pol II leaves the promoter and carries out chain elongation. Interestingly, the same general transcription factors, including TFIID, bind to the TATA-less promoter, even though TATA binding by TBP is not available.
   
   
4. A unique difference between prokaryotic and eukaryotic transcription is that in prokaryotes a singlemRNA containing many genes can be transcribed from the DNA template as a single transcription unit, coupled with their direct translation on ribosomes into discrete polypeptides. This process reflects the fact that genes which encode enzymes in a given pathway are often clustered in an operon and are co-ordinately regulated.
   
   In contrast to the synthesis of polycistronic mRNA in E. coli and other bacteria, eukaryotic transcription units usually consists of single genes. This characteristic may also reflect uncoupled transcription and translation in these organisms. Thus, heterogeneous nuclear RNA (hnRNA) is synthesized in the nucleus and then transported to the cytoplasm along with its processing into mature mRNA including splicing, addition of poly(A) tail at the 3´ end, and capping at the 5´ end. Subsequently, the RNA is translated on ribosomes (endoplasmic reticulum). Thus, synthesis and utilization of mRNA are temporally and spatially separated.