Transcription eukaryotic promoters

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

General transcription factors (GTFs) that bind to eukaryotic promoters are functionally analogous to a factor in prokaryotes. 

The sequences of eukaryotic promoters are more variable than their prokaryotic counterparts (see Fig. 26-8). The three eukaryotic RNA polymerases usually require an array of general transcription factors in order to bind to a promoter. Yet, as with prokaryotic gene expression, the basal level of transcription is determined by the effect of promoter sequences on the function of RNA polymerase and its associated transcription factors. 

A major goal in recombinant DNA technology is the production of useful foreign proteins by bacteria, yeast, or other cultured cells. Protein synthesis depends upon both transcription and translation of the cloned genes and may also involve secretion of proteins from the host cells. The first step, transcription, is controlled to a major extent by the structures of promoters and other control elements in the DNA (Chapter 28). Since eukaryotic promoters often function poorly in bacteria, it is customary to put the cloned gene under the control of a strong bacterial or viral X promoter. The latter include the X promoter PL (Fig. 28-8) and the lac (Fig. 28-2) and trp promoters of E. coli. These are all available in cloning vehicles. 

Phil Sharp and Leonard Guarente showed that at least four transcription factors are required in addition to polymerase II for initiation from the major late promoter of adenovirus. In vitro studies indicate that these factors assemble in an orderly fashion (see fig. 28.12b). First the TFIID complex binds to the TATA box. Sequential binding of TFIIA, TFIIB, RNA polymerase II, and TFIIE follow. It is believed that this multifactor complex functions for a large number of eukaryotic promoters that contain TATA boxes. 

Allele-specific differences between regulatory polymorphisms associated with the ability of RNA polymerase II to bind and assemble its transcription complex at the start site of transcription for several eukaryotic promoters has also been measured using MALDI-TOF MS coupled with primer extension (42). This technique provides a powerful tool for identifying important regulatory SNPs and haplotypes in vivo. 

RNA polymerase II transcribes messenger RNA and a few other small cellular RNAs. Class II promoters are usually defined by their sensitivity to a-amanitin. Like prokaryotic promoters, many class II promoters contain two conserved sequences, called the CAAT and TATA boxes. The TATA box is bound by a specialized transcription factor called TBP (for TATA-Binding-Factor). Binding of TBP is required for transcription, but other proteins are required to bind to the upstream (and potentially downstream) sequences that are specific to each gene. Like prokaryotic transcripts, eukaryotic RNAs are initiated with a nucleoside triphosphate. Termination of eukaryotic mRNA transcription is less well understood than is termination of prokaryotic transcription, because the 3 ends of eukaryotic mRNAs are derived by processing. See Figure 12-9. 

The tasks of transcriptional and translational signal recognition involve the prediction of promoters and sites that function in the initiation and termination of transcription and translation. Bacterial promoter sites, specifically the Escherichia coli RNA polymerase promoter site, are now very well characterized. The main problem is that the two conserved regions of the bacterial promoter, the -10 and -35 regions, are separated from each other by 15 to 21 bases, making the detection of the entire promoter as a single pattern difficult. Eukaryotic promoters are less well characterized than their bacterial equivalents. The major elements are the CCAAT box, GC box, TATA box and cap site. 

Eukaryotic genes encoding proteins have promoter sites with a TATAAA consensus sequence, called a TATA box or a Hogness box, centered at about -25 (Figure 5.27B). Many eukaryotic promoters also have a CAATbox with a GGNCT4TCT consensus sequence centered at about -75. Transcription of eukaryotic genes is further stimulated by... 

Multiple Transcription Factors Interact with Eukaryotic Promoters... 

The structure of eukaryotic promoters is more complex than that of prokaryotic promoters. DNA sequences, hundreds of base pairs (bp) upstream from the transcription start site, control the rate of initiation. Furthermore, initiation requires numerous specific proteins (transcription factors) that bind to particular DNA sequences. Without the transcription factors, RNA polymerase II cannot bind to a promoter. However, KNH polymerase II itself is not a transcription factor. The complexity of initiation may derive, in part, from the fact that eukaryotic DNA is in the form of chromatin, which is inaccessible to RNA polymerases. Many RNA polymerase II promoters have the following features ... 

Eukaryotic genes, like prokaryotic genes, require promoters for transcription initiation. Like prokaryotic promoters, eukaryotic promoters consist of conserved sequences that serve to attract the polymerase to the start site. However, eukaryotic promoters differ distinctly in sequence and position, depending on the type of RNA polymerase to which they hind (Figure 29.17). 

Figure 29.17 Common eukaryotic promoter elements. Each eukaryotic RNA polymerase recognizes a set of promoier elements—sequences in DNA that promote transcription 1 he KNA polymerase I promoter consists of a ribosomal initator (rlnr) and an upstream promoter element (UPE). Tfie RNA polymerase II promotei likewise includes an initator element (Inr) and may also include either a TATA box or a downstream promoter dement (DPE), Separate from the promoter region, enhancer dements bind specific transcription factors. RNA polymerase III promoters consist of conserved sequences that lie within the transcribed genes.

Kondrakhin, Y. V., Kel, A. E., Kolchanov, N. A., Romashchenko, A. G., Miianesi, L. (1995) Eukaryotic promoter recognition by binding sites for transcription factors. Comp Appl Biosci 11, 477-488. 

The first and most well-known promoter database, the Eukaryotic Promoter Database (EPD) (I) consists of promoter sequences extracted from the up- and downstream regions of either experimentally or in. vt/firt-determined transcription... 

A prototypical RNA pol II-dependent eukaryotic promoter. Three classes of transcription factors are schematically represented the TFII auxiliary factors (TFIIF, TFIIJ, TFIIH, etc.), TBP with its associated factors (TAFs), and various transcriptional regulatory factors which bind to DNA sequences called response elements (RE). See Chapter 24 for more information about TBP and the RNA pol II holoenzyme. 

Initiation complex - All of the protein-coding genes in eukaryotes are transcribed by RNA polymerase II (pol II) This enzyme also transcribes some of the small nuclear RNAs involved in splicing (see here). Like other RNA polymerases, pol II is a complex, multisubunit enzyme, but not even its numerous subunits are sufficient to allow pol II to initiate transcription on a eukaryotic promoter. To form a minimal complex capable of initiation, at least five additional protein factors are needed Figure 28.24 and listed in Table 28.4. The minimal unit involves the TATA binding protein, (TBP), but in vivo formation of the complex probably always uses TFllD, a multi-subunit structure incorporating both TBP and TATA binding associated factors (TAFs). 

Fig. 14.5. Prokaryotic and eukaryotic promoters. The promoter-proximal region contains binding sites for transcription factors which that can accelerate the rate at which RNA polymerase binds to the promoter. Pu = purine Py = pyrimidine.

TFIID is one of the general transcription factors for RNA polymerase II. Part of it is a protein that binds to the TATA box in eukaryotic promoters. Associated in complex with the TATA box and the TBP are many proteins called TAFs, for TBP associated factors. 

---