Transcription, eucaryotic

The specific arrangement of two a helices joined by a loop region in lambda Cro and repressor, as well as in CAP, constitute the helix-turn-helix DNA-binding motif (Figure 8.8), which also occurs in some eucaryotic transcription factors as discussed in Chapter 9. The orientation of the two helices and... 

Figure 9.1 The transcriptional elements of a eucaryotic structural gene extend over a large region of DNA. The regulatory sequences can be divided into three main regions (1) the basal promoter elements such as the TATA box, (2) the promoter proximal elements close to the initiation site, and (3) distal enhancer elements far from the initiation site.

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

The two homologous repeats, each of 88 amino acids, at both ends of the TBP DNA-binding domain form two stmcturally very similar motifs. The two motifs each comprise an antiparallel p sheet of five strands and two helices (Figure 9.4). These two motifs are joined together by a short loop to make a 10-stranded p sheet which forms a saddle-shaped molecule. The loops that connect p strands 2 and 3 of each motif can be visualized as the stirmps of this molecular saddle. The underside of the saddle forms a concave surface built up by the central eight strands of the p sheet (see Figure 9.4a). Side chains from this side of the P sheet, as well as residues from the stirrups, form the DNA-binding site. No a helices are involved in the interaction area, in contrast to the situation in most other eucaryotic transcription factors (see below). 

Eucaryotes have many more genes and a broader range of specific transcription factors than procaryotes and gene expression is regulated by using sets of these factors in a combinatorial way. Eucaryotes have found several different solutions to the problem of producing a three-dimensional scaffold that allows a protein to interact specifically with DNA. In the next chapter we shall discuss some of the solutions that have no counterpart in procaryotes. However, the procaryotic helix-turn-helix solution to this problem (see Chapter 8) is also exploited in eucaryotes, in homeodomain proteins and some other families of transcription factors. 

Goodrich, J.A., Tjian, R. TBP TAF complexes selectivity factors for eucaryotic transcription. Curr. Opin. Cell Biol. 6 403-409, 1994. 

Leucine zippers provide dimerization interactions for some eucaryotic transcription factors... 

Bacteria, being procaryotic, do not show compartmentation of the biosynthetic processes. The genome of a bacterium relates directly to the cytoplasm of the cell. Transcription into mRNA can lead directly to translation, and the processes of transcription and translation are not carried out in separate organelles. Animal cells, being eucaryotic, show compartmentation of the transcription and translation processes. Transcription of the genome into mRNA occurs in the nucleus, whereas translation occurs in the cytoplasm. The messenger RNA in the eucaryote is usually modified by adding to it... 

In vitro studies of transcription on nucleosomes 3.1. The eucaryotic polymerases... 

The first zinc binding motif discovered was that of the eucaryotic transcription factor TFIIIA of Xenopus laevis which contains 9 copies of a Cys2His2-Zinc motif The structure of the binding motif is shown in Fig. 1.4. The central zinc ion serves to pack an a-hehx against a P-sheet and thereby position the a-helix. The recognition of the DNA sequence occurs via this a-helix. 

Fig. 1.7. Basic leudne zipper and heltx-loop-heltx motif in complex with DNA. A) The basic leucine zipper of the transcription activator GCN4 of yeast consists of two slightly curved a-hehces, which dimerize with the help of the leucine zipper motif. The sequence specific binding of DNA occurs via the basic ends of the two helices. They insert themselves into the major groove of the DNA. B) The helix-loop-helix motif of the eucaryotic transcription factor Max complexed with DNA. Molscript drawing (Kraulis 1991).

Fig. 1.10. The eucaryotic transcription factor NFxB in complex with DNA. Shown is the structure of a fragment of the p50 subunit of NFxB complexed with the recognition sequence. p50 of NFxB binds DNA as a dimer. Each of the subunits contains a bundle of P-sheets which envelops the DNA so that only the minor groove is exposed. After Ghosh et al. (1995), with permission.

Cis-acting DNA elements can he near the start site of transcription or be quite distanced from it. Fmthermore, there are examples among eucaryotes in which the cis element is foimd within the transcribed region. If the cis element is located far from the site of action and its effect is also orientation-independent, then it is termed an enhancer. Fmthermore, one frequently observes in eucaryotes so called composite control regions which contain various cis elements. In this case, several transcription factors act cooperatively in the initiation of transcription. Examples for such cooperative effects are observed among the genes controlled by nuclear receptors. 

Of particular importance is the phosphorylation of eucaryotic transcription factors. Functional and mechanistic consequences of the phosphorylation of transcription factors will be discussed in more detail in the section on the regulation of eucaryotic transcription (see 1.4.3.2). Specific or non-specific protein phosphatases (see 7.5) can remove the phosphate residues and terminate the phosphorylation signal. 

The amoimt of available DNA-binding proteins is, in many situations, a critical factor for the extent of transcription regulation. The concentration of regulatory DNA-binding proteins can be regulated within the framework of the following processes in eucaryotes ... 

Procaryotes and eucaryotes differ decisively in the structure of the transcription start site and the complexity of the transcription appartus. For a better understanding we want to briefly summarize procaryotic transcription and then contrast it to eucaryotic transcription (review Eick and Heumarm, 1994). 

The RNA polymerase of E. coli possesses with its subimit construction (a2PP o) a simple structure in comparison to eucaryotic RNA polymerases. The sigma factor is only required for the recognition of the promoter and the subsequent formation of a tight complex. After the incorporation of the first 8-10 nucleotides into the transcript, the sigma factor dissociates from the holoenzyme, and the remaining core enzyme carries out the rest of the elongation. 

A comparison of the activation of and o " -dependent promoters helps us understand some of the basic points of transcription activation, which also play an important role in eucaryotic transcription. 

Tliree types of RNA polymerases exist for the transcription of eucaryotic genes, each of which transcribes a certain class of genes. All three enzymes are characterized by a complex subunit structure. 

A combination of several cis-elements, and thus several transcriptional activators, are often involved in the regulation of eucaryotic transcription. Transcription activation, in these cases, results from the complex concerted action of various specific DNA-binding proteins. 

Fig. 1.30. Structure of a typical eucaryotic transcription start site. Enhancer elements and UAS elements (UAS upstream activating sequences) are binding sites for positive and negative regulatory DNA-binding proteins. The TATA box is the binding site for the TATA box binding protein (TBP) and serves to position the RNA polymerase holoenzyme on the promoter. For promoters that do not possess a TATA box, this function is fulfilled by an initiator region.

As in procaryotes, the elementary steps of initiation, elongation and termination can be distinguished in eucaryotic transcription. Aside from the specific RNA polymerases, transcription in eucaryotes requires the action of numerous other proteins which are collectively known as transcription factors. Transcription factors are required at the level of initiation, elongation, and termination and are accordingly known as initiation factors, elongation factors and termination factors of transcription. 

Transcription in eucaryotes can, as shown schematically in Fig. 1.31, be subdivided in the following steps (Review Roeder, 1996, Nikolov and Burley, 1997) ... 

In contrast to the procaryotes, where the o -holoenzyme of the RNA polymerase can initiate transcription without the aid of accessory factors, the eucaryotic RNA polymerase requires the help of numerous proteins to begin transcription. These proteins are termed basal or general initiation factors of transcription. Together with RNA polymerase II, they participate in the basal transcription apparatus. The various components must associate in a defined order for the formation of a transcription-competent complex, from which a low level of transcription is possible. An increase in the basal transcriptional level requires the effect of specific transcriptional activators, which bind cognate DNA sequences at a variable distance from the promoter. The transcriptional activators themselves require the aid of further protein factors, known as coactivators (see 1.4.3.2), in order to attain full stimulatory activity. 

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