Promoters eukaryotic

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). 

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). 

Consensus sequence in the promoter region of many eukaryotic genes that bind a general transcription factor and hence specifies the position where transcription is initiated by the RNA polymerase. 

It is clear that the signals in DNA which control transcription in eukaryotic cells are of several types. Two types of sequence elements are promoter-proximal. One of these defines where transcription is to commence... 

A third class of sequence elements can either increase or decrease the rate of transcription initiation of eukaryotic genes. These elements are called either enhancers or repressors (or silencers), depending on which effect they have. They have been found in a variety of locations both upstream and downstream of the transcription start site and even within the transcribed portions of some genes. In contrast to proximal and upstream promoter elements, enhancers and silencers can exert their effects when located hundreds or even thousands of bases away from transcription units located on the same chromosome. Surprisingly, enhancers and silencers can function in an orientation-independent fashion. Literally hundreds of these elements have been described. In some cases, the sequence requirements for binding are rigidly constrained in others, considerable sequence variation is... 

Figure 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions. The entire complex spans DNA from position -30 to +30 relative to the initiation site (+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and ofTBP bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 A resolution. The structure of TFIID complexes have been determined by electron microscopy at 30 A resolution. Thus, the molecular structures of the transcription machinery are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

RNA polymerases interact with unique cw-active regions of genes, termed promoters, in order to form preinitiation complexes (PICs) capable of initiation. In eukaryotes the process of PIC formation is facilitated by multiple general transcription factors (GTFs), TFIIA, B, D, E, F, and H. 

In addition to affecting the efficiency of promoter utilization, eukaryotic cells employ alternative RNA processing to control gene expression. This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used. Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differendy in different tissues. A few examples of each of these types of regulation are presented below. 

The structure and sequence of the catalytic moiety have been determined (06, V6, W6). The enzyme consists of 362 amino acids and 40,638 daltons of the protein predicted by the cDNA sequence. The ADA gene spans 32 kb and consists of 12 exons. The apparent promoter region of the gene lacks the TATA and CAAT sequences often found in eukaryotic promoters and is extremely G/C rich. The location of the ADA gene is on chromosome 20ql2-ql3.11 (Jl). 

Gomez, E., and Pavitt, G. D. (2000). Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation. Mol. Cell Biol. 20, 3965—3976. 

Nielsen, K. H., Valasek, L., Sykes, C., Jivotovskaya, A., and Hinnebusch, A. G. (2006). Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast. Mol. Cell. Biol. 26, 2984—2998. 

Genes of interest can be tagged at either the N or C terminal end. The decision to tag a protein at either the N or the C terminal depends upon the properties of the protein of interest. In our case, all the eukaryotic translation initiation factors were tagged C terminally to allow the endogenous promoter to influence the expression of the tagged protein. 

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