Transcription start sites

The DNA part of each control module can be divided into three main regions, the core or basal promoter elements, the promoter proximal elements and the distal enhancer elements (Figure 9.1). The best characterized core promoter element is the TATA box, a DNA sequence that is rich in A-T base pairs and located 25 base pairs upstream of the transcription start site. The TATA box is recognized by one of the basal transcription factors, the TATA box-binding protein, TBP, which is part of a multisubunit complex called TFIID. This complex in combination with RNA polymerase 11 and other basal transcription factors such as TFIIA and TFIIB form a preinitiation complex for transcription. 

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. 

General or basic transcription factors are required for every gene to allow the proper recruitment of RNA polymerases to ensure transcriptional activity. They bind to core promoters in the vicinity of transcriptional start sites in a sequential manner. 

Proteins that bind DNA at specific DNA sequences often distal from transcriptional start sites of genes. Their binding and activity is usually cell type or stimulus triggered, which subsequently decondensate the chromatin and finally lead to the recruitment of general transcription factors and the RNA polymerase. 

A sequence stretch 300 base pairs upstream of the transcriptional start site suffices for most of the transcriptional regulation of the IL-6 gene (Fig. 1). Within this sequence stretch several transcription factors find their specific recognition sites. In 5 to 3 direction, AP-1, CREB, C/EBP 3/NF-IL6, SP-1 and NF-kB can bind to the promoter followed by TATA and its TATA binding protein TBP. Most enhancer factors become active in response to one or several different stimuli and the active factors can trigger transcription individually or in concert. For example, AP-1 is active upon cellular stress, or upon stimuli that tell cells to proliferate CREB becomes also active if cells experience growth signals, but also upon elevation of intracellular levels of cyclic adenosine monophosphate (cAMP), which occurs upon stimulation if so called hormone-activated G protein-coupled receptors. 

Indicates the mapped potential putative transcription start site. The cold box Is shown In boldface. 

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... 

The use of alternative transcription start sites results in a different 5 exon on mRNAs corresponding to... 

The transactivation-responsive region (TAR) is located immediately downstream of the transcriptional start site in the HIV-1 LTR, encompassing nucleotides h-1 to h-59 (Berkhout et al. 1989 Muesing et al. 1987), and is reqnired for the function of the viral trans-activator protein Tat. In the absence of Tat (Fig. 5.2b), short transcripts... 

The highly restricted tissue-specific transcription of c-mos poses an interesting problem in gene regulation during germ cell development. Moreover, c-mos is transcribed from different promoters in mouse spermatocytes and oocytes (Fig. 4) (Propst et al., 1987). In spermatocytes, transcription initiates approximately 280 nucleotides upstream of the c-mos ATG (Propst et al., 1987), whereas the transcription start site in oocytes has been mapped to 53 base pairs upstream of the ATG (Pal et al., 1991). Neither the spermatocyte nor oocyte promoter regions are... 

Figure 4. Transcriptional regulatory sequences of the mouse c-mos gene. Transcription in oocytes and spermatocytes initiates 53 and approximately 280 base pairs upstream of the c-mos ATG, respectively. Efficient transcription in oocytes requires an initiator (Inr)-I ike sequence located downstream of the transcription start site. A negative regulatory element (NRE) located upstream of the spermatocyte promoter acts to suppress c-mos transcription in somatic cells.

Figure 6. The c-mos negative regulatory element (NRE). Nucleotide positions of the NRE are shown relative to the spermatocyte transcription start site, taken as 280 base pairs upstream of the c-mos ATG (see Fig. 4). The endpoints of the NRE are defined by deletions that allow c-mos expression in NIH 3T3 and other somatic cells. Mutations of the sequences designated by boxes 1,2, and 3 also allow c-mos transcription in NIH 3T3 cells, indicating that these sequences represent functional elements within the NRE. Boxes 1 and 2 are similar to sequences upstream of the protamine (Prot) promoter that inhibit in vitro transcription in HeLa cell extracts. A sequence just upstream of box 2 is also similar to a putative repressor-binding site in the regulatory region of Pgk2.

The transcription factor Spl plays the predominant role in the expression of ADH5 [31, 33]. There is a pair of Spl sites flanking the transcription start site, and a minimal promoter containing these sites is a strong promoter in several different cell types [31]. This region of the promoter is essentially inactive in Drosophila cells that lack Spl, and are strongly activated by coexpression of Spl [33]. 

The first step in creating a specific mRNA species is to construct a plasmid encoding the mRNA behind a T7, T3, or SP6 RNA polymerase promoter sequence. The S boundary of the mRNA is defined by the position of the promoter transcription start site and the S end is defined by restriction digest of the plasmid prior to in vitro transcription. 

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