Promoter, enhancer & silencer sites for initiation of transcription in eukaryotes

Content
Expression of Gene : Protein Synthesis 2.  Transcription in Prokaryotes and Eukaryotes
Transcription in prokaryotes 
Single RNA polymerase in E. coli
Promoter sites for initiation of transcription in prokaryotes
Initiation and elongation of RNA synthesis in prokaryotes
'Inchworm model' for elongation of transcript
Elongation arrest vs termination of transcription
Termination and antitermination of mRNA synthesis in prokaryotes
Transcription in eukaryotes 
Multiple RNA polymerases in eukaryotes
Promoter, enhancer and silencer sites for initiation of transcription in eukaryotes
Transcription factors and initiation of RNA synthesis in eukaryotes
Formation of preinitiation (transcription) complex with RNA polymerase II (Pol II)
Structure and role of TFIID and other transcription factors (TBP, TAFs)
TFIIB domains for interaction with TFIID/TATA complex
Phosphorylation of CTD of a subunit of Pol II
Formation of pre-initiation complex with Pol I and Pol III
Separate DNA binding and transcription activation domains
Transcription factors and elongation of RNA chains in eukaryotes
Chromatin structure and transcription
Transcription in mitochondria
Transcription of vertebrate mtDNA
Transcription of yeast and plant mtDNA
Transcription in chloroplasts
Promoter, enhancer and silencer sites for initiation of transcription in eukaryotes
As earlier discussed, in eukaryotes there are three RNA polymerases (RNA polymerase I for rRNA; RNA polymerase II for hnRNA and RNA polymerase III for 5S RNA, tRNAs, U6 snRNA, 7SK, EBER2). Promoters for RNA polymerase I could not be initially studied (although recently promoters for this enzyme for rRNA have been studied and their properties described), since all genes for rRNA were similar. Promoters for RNA polymerase III, on the other hand, had some unusual downstream promoters (to be discussed later). However, for RNA polymerase II, several hundred eukaryotic genes have now been sequenced and their promoters studied revealing some general features in three regions located at start point, centred at sites lying between -25 bp and -100 bp. The least effective of these three regions is the TATA or Hogness box (7 bp long) located 20 bp upstream to the start point. The TATA or Hogness box has the following sequence (frequency shown by subscripts).



The TATA box is surrounded by G-C rich sequences and is comparable to Pribnow box of prokaryotes (see Regulation of Gene Expression 3. A Variety of Mechanisms in Eukaryotes also). Further upstream is another sequence called CAAT box, which, being necessary for initiation, is conserved in some promoters (β globin gene), but is not necessary in some other genes (thymidine kinase gene of herpes virus). This sequence lies between -70 and -80 base pairs and includes the following.


Another sequence called GC box (GGGC GG) is found in one or more copies at -60 or -100 bp upstream in any orientation in several genes. It has been shown that CAAT and GC boxes determine the efficiency of transcription, while TATA box aligns RNA polymerase at proper site, with the help of TFIID and other transcription factors. (See later for transcription factors).

Eukaryotic promoters often also possess elements located 100 or 200 base pairs upstream, which interact with proteins other than RNA polymerase and thus regulate the activity of promoter. These elements, called enhancers, also called upstream activation sites or UAS, can be moved several hundred or even thousands of base pairs upstrearrj,or downstream without alteration of their activities. These enhancers may lead upto a 200 fold increase in transcription rate of an affected gene, hence the name. There are other regulatory elements known as silencers, which repress gene expression. As shown by Alexander Johnson (San Francisco), silencers, like enhancers,can function at great distances from genes they repress. They are sites for binding of proteins just like enhancers. The location of different promoters and enhancers is shown in Fig. 32.13. More details about enhancers and their role in gene expression are given in Regulation of Gene Expression 3. A Variety of Mechanisms in Eukaryotes. (Silencers are found in yeast and repress expression of HML and HMR loci involved in switching of mating types; consult Regulation of Gene Expression 3. A Variety of Mechanisms in Eukaryotes).


A DNA segment rn a eukaryote showing promoter sites (TATA, GC and CAAT boxes) and the enhancer site.
Fig. 32.13. A DNA segment rn a eukaryote showing promoter sites (TATA, GC and CAAT boxes) and the enhancer site.

In 5SRNA genes transcribed by RNA polymerase III, the promoter lies well within the transcription unit, 50 bp downstream rather than upstream from the start point (Fig. 32.14). 5SRNA keeps on being synthesized even when the entire sequence upstream from the gene is removed suggesting that no upstream promoter is involved in transcription. However, deletions of bases downstream from start point, if lie between 55—80 bp lead to complete halt of transcription. Therefore, 50-80bp sequence downstream in 5SRNA gene constitutes the promoter region. The location of downstream promoters for RNA polymerase III .may vary in different genes (Fig. 32.14).
 
Three different genes transcribed by RNA polymerase III, all containing internal promoters; numbers indicate the number of bases from the start point of the gene.
Fig. 32.14. Three different genes transcribed by RNA polymerase III, all containing internal promoters; numbers indicate the number of bases from the start point of the gene.

One would be interested in knowing how RNApolymerase III, attached to +55 to +80 bp sequence, can start transcription from the start point. It is believed that the enzyme is big enough to occupy start point and the promoter region simultaneously and thus may start transcribing at the start point without difficulty.

The downstream promoter sequences of 5S RNA and tRNA genes have subsequently been delineated more precisely into box A, box B and box C In 5S genes box A and box C are found at +50 to +69 and +80 to +90 respectively. Similarly in tRNA genes, box A and box B are found at +8 to +30 and +51 to +72 respectively. It has also been shown, that box A in 5S genes and tRNA genes, though differs in location, but has similar ; conserved sequences'. It is, therefore, recognised by the same transcription factor (TFIIIB). These promoter sequences and the transcription factors binding at these sites are shown in Figure 32.15 (see later for more details).

Some RNA polymerase III genes are also known to have upstream promoters like TATA box (for example U6 snRNA, 7SK, EBER2; genes for other snRNAs, like U2, are transcribed by RNA polymerase II). Among these genes 7S genes have both types of promoters (downstream and upstream), so that on deletion of box A, upstream promoter is used by RNA polymerase III.
 
Binding of TFIIIB to downstream promoters (box A), located at different positions in 5S and tRNA genes.
Fig. 32.15. Binding of TFIIIB to downstream promoters (box A), located at different positions in 5S and tRNA genes.