Harpers

350 / CHAPTER 37Finally, this newly formed 3′ terminal is polyadenylatedin the nucleoplasm, as described below.THE EUKARYOTICTRANSCRIPTION COMPLEXA complex apparatus consisting of as many as 50unique proteins provides accurate and regulatable transcriptionof eukaryotic genes. The RNA polymerase enzymes(pol I, pol II, and pol III for class I, II, and IIIgenes, respectively) transcribe information contained inthe template strand of DNA into RNA. These polymerasesmust recognize a specific site in the promoter inorder to initiate transcription at the proper nucleotide.In contrast to the situation in prokaryotes, eukaryoticRNA polymerases alone are not able to discriminate betweenpromoter sequences and other regions of DNA;thus, other proteins known as general transcription factorsor GTFs facilitate promoter-specific binding ofthese enzymes and formation of the preinitiation complex(PIC). This combination of components can catalyzebasal or (non)-unregulated transcription in vitro.Another set of proteins—coactivators—help regulatethe rate of transcription initiation by interacting withtranscription activators that bind to upstream DNA elements(see below).Formation of the BasalTranscription ComplexIn bacteria, a σ factor–polymerase complex selectivelybinds to DNA in the promoter forming the PIC. Thesituation is more complex in eukaryotic genes. Class IIgenes—those transcribed by pol II to make mRNA—are described as an example. In class II genes, the functionof σ factors is assumed by a number of proteins.Basal transcription requires, in addition to pol II, anumber of GTFs called TFIIA, TFIIB, TFIID,TFIIE, TFIIF, and TFIIH. These GTFs serve to promoteRNA polymerase II transcription on essentially allgenes. Some of these GTFs are composed of multiplesubunits. TFIID, which binds to the TATA box promoterelement, is the only one of these factors capableof binding to specific sequences of DNA. As describedabove, TFIID consists of TATA bindingprotein (TBP) and 14 TBP-associated factors (TAFs).TBP binds to the TATA box in the minor groove ofDNA (most transcription factors bind in the majorgroove) and causes an approximately 100-degree bendor kink of the DNA helix. This bending is thought tofacilitate the interaction of TBP-associated factors withother components of the transcription initiation complexand possibly with factors bound to upstream elements.Although defined as a component of class IIgene promoters, TBP, by virtue of its association withdistinct, polymerase-specific sets of TAFs, is also an importantcomponent of class I and class III initiationcomplexes even if they do not contain TATA boxes.The binding of TBP marks a specific promoter fortranscription and is the only step in the assembly processthat is entirely dependent on specific, high-affinity protein-DNAinteraction. Of several subsequent in vitrosteps, the first is the binding of TFIIB to the TFIIDpromotercomplex. This results in a stable ternary complexwhich is then more precisely located and moretightly bound at the transcription initiation site. Thiscomplex then attracts and tethers the pol II-TFIIF complexto the promoter. TFIIF is structurally and functionallysimilar to the bacterial σ factor and is requiredfor the delivery of pol II to the promoter. TFIIA bindsto this assembly and may allow the complex to respondto activators, perhaps by the displacement of repressors.Addition of TFIIE and TFIIH is the final step in the assemblyof the PIC. TFIIE appears to join the complexwith pol II-TFIIF, and TFIIH is then recruited. Each ofthese binding events extends the size of the complex sothat finally about 60 bp (from −30 to +30 relative to +1,the nucleotide from which transcription commences)are covered (Figure 37–9). The PIC is now completeand capable of basal transcription initiated from the correctnucleotide. In genes that lack a TATA box, thesame factors, including TBP, are required. In such cases,an Inr or the DPEs (see Figure 37–8) position the complexfor accurate initiation of transcription.Phosphorylation Activates Pol IIEukaryotic pol II consists of 12 subunits. The twolargest subunits, both about 200 kDa, are homologousto the bacterial β and β′ subunits. In addition to the increasednumber of subunits, eukaryotic pol II differsfrom its prokaryotic counterpart in that it has a series ofheptad repeats with consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at the carboxyl terminal of the largestpol II subunit. This carboxyl terminal repeat domain(CTD) has 26 repeated units in brewers’ yeast and 52units in mammalian cells. The CTD is both a substratefor several kinases, including the kinase component ofTFIIH, and a binding site for a wide array of proteins.The CTD has been shown to interact with RNA processingenzymes; such binding may be involved withRNA polyadenylation. The association of the factorswith the CTD of RNA polymerase II (and other componentsof the basal machinery) somehow serves tocouple initiation with mRNA 3′ end formation. Pol IIis activated when phosphorylated on the Ser and Thrresidues and displays reduced activity when the CTD isdephosphorylated. Pol II lacking the CTD tail is incapableof activating transcription, which underscores theimportance of this domain.