DNA-Dependent Synthesis of RNA: -RNA Polymerase II Requires Many Other Protein Factors for Its Activity
RNA polymerase II is central to eukaryotic gene expression and has been studied extensively. Although this polymerase is strikingly more complex than its bacterial counterpart, the complexity masks a remarkable conservation of structure, function, and mechanism. Pol II is a huge enzyme with 12 subunits. The largest subunit (RBP1) exhibits a high degree of homology to the β subunit of bacterial RNA polymerase. Another subunit (RBP2) is structurally similar to the bacterial subunit, and
others (RBP3 and RBP11) show some structural homology to the two bacterial α subunits. Pol II must function with genomes that are more complex and with DNA molecules more elaborately packaged than in bacteria. The need for protein-protein contacts with the numerous other protein factors required to navigate this labyrinth accounts in large measure for the added complexity of the eukaryotic polymerase.
The largest subunit of Pol II also has an unusual feature, a long carboxyl-terminal tail consisting of many re peats of a consensus heptad amino acid sequence–YSPTSPS–. There are 27 repeats in the yeast enzyme (18 exactly matching the consensus) and 52 (21 exact) in the mouse and human enzymes. This carboxyl terminal domain (CTD) is separated from the main body of the enzyme by an unstructured linker sequence. The CTD has many important roles in Pol II function, as out lined below.
RNA polymerase II requires an array of other proteins, called transcription factors, in order to form the active transcription complex. The general transcription factors required at every Pol II promoter
(factors usually designated TFII with an additional identifier) are highly conserved in all eukaryotes (Table 26–1). The process of transcription by Pol II can be described in terms of several phases—assembly, initiation, elongation, termination—each associated with characteristic proteins (Fig. 1). The step-by-step pathway described below leads to active transcription in vitro. In the cell, many of the proteins may be present in larger, preassembled complexes, simplifying the pathways for assembly on promoters. As you read about this process.
FIGURE1 Transcription at RNA polymerase II promoters. (a) The sequential assembly of TBP (often with TFIIA), TFIIB, TFIIF plus Pol II, TFIIE, and TFIIH results in a closed complex. TBP often binds as part of a larger complex, TFIID. Some of the TFIID subunits play a role in transcription regulation . Within the complex, the DNA is unwound at the Inr region by the helicase activity of TFIIH and perhaps of TFIIE, creating an open complex. The carboxyl-terminal domain of the largest Pol II subunit is phosphorylated by TFIIH, and the polymerase then escapes the promoter and begins transcription. Elon gation is accompanied by the release of many transcription factors and is also enhanced by elongation factors . After termination, Pol II is released, dephosphorylated, and recycled. (b) The structure of human TBP (gray) bound to DNA (blue and white) (PDB ID 1TGH).
Assembly of RNA Polymerase and Transcription Factors at a Promoter The formation of a closed complex begins when the TATA-binding protein (TBP) binds to the TATA box . TBP is bound in turn by the transcription factor TFIIB, which also binds to DNA on either side of TBP. TFIIA binding, although not always essential, can stabilize the TFIIB-TBP complex on the DNA and can be important at nonconsensus promoters where TBP binding is relatively weak. The TFIIB-TBP complex is next bound by another complex consisting of TFIIF and Pol II. TFIIF helps target Pol II to its promoters, both by interacting with TFIIB and by reducing the binding of the polymerase to nonspecific sites on the DNA. Finally, TFIIE and TFIIH bind to create the closed complex. TFIIH has DNA helicase activity that promotes the unwinding of DNA near the RNA start site (a process requiring the hydrolysis of ATP), thereby creating an open complex. Counting all the subunits of the various essential factors (excluding TFIIA), this mini mal active assembly has more than 30 polypeptides.
RNA Strand Initiation and Promoter Clearance TFIIH has an additional function during the initiation phase. A kinase activity in one of its subunits phosphorylates Pol II at many places in the CTD. Several other protein kinases, including CDK9 (cyclin-dependent kinase 9), which is part of the complex pTEFb (positive transcription elongation factor b), also phosphorylate the CTD. This causes a conformational change in the overall complex, initiating transcription. Phosphorylation of the CTD is also important during the subsequent elongation phase, and it affects the interactions between the transcription complex and other enzymes involved in processing the transcript (as described below). During synthesis of the initial 60 to 70 nucleotides of RNA, first TFIIE and then TFIIH is released, and Pol II enters the elongation phase of transcription.
Elongation, Termination, and Release TFIIF remains associated with Pol II throughout elongation. During this stage, the activity of the polymerase is greatly enhanced by proteins called elongation factors (Table 26–1). The elongation factors suppress pausing during transcription and also coordinate interactions between protein complexes involved in the posttranscriptional processing of mRNAs. Once the RNA transcript is completed, transcription is terminated. Pol II is dephosphorylated and recycled, ready to initiate another transcript .
Regulation of RNA Polymerase II Activity Regulation of transcription at Pol II promoters is quite elaborate. It in volves the interaction of a wide variety of other proteins with the preinitiation complex. Some of these regulatory proteins interact with transcription factors, others with Pol II itself. Many interact through TFIID, a com plex of about 12 proteins, including TBP and certain TBP-associated factors, or TAFs.
Diverse Functions of TFIIH In eukaryotes, the repair of damaged DNA is more efficient within genes that are actively being transcribed than for other damaged DNA, and the template strand is repaired somewhat more efficiently than the nontemplate strand. These remarkable observations are explained by the alternative roles of the TFIIH subunits. Not only does TFIIH participate in the formation of the closed com plex during assembly of a transcription complex (as described above), but some of its subunits are also essential components of the separate nucleotide-excision repair complex . When Pol II transcription halts at the site of a DNA lesion, TFIIH can interact with the lesion and recruit the entire nucleotide-excision repair com plex. Genetic loss of certain TFIIH subunits can produce human diseases. Some examples are xeroderma pigmentosum and Cockayne’s syndrome, which is characterized by arrested growth, photosensitivity, and neurological disorders
