Transcription Initiation by Pol I and Pol III Is Analogous to That by Pol II

The formation of transcription initiation complexes involving Pol I and Pol III is similar in some respects to assembly of Pol II initiation complexes (see Figure 9-19). However, each of the three eukaryotic nuclear RNA polymerases requires its own polymerase-specific general transcription factors and recognizes different DNA control elements. Moreover, neither Pol I nor Pol III requires ATP hydrolysis by a DNA helicase to help melt the DNA template strands to initiate transcription, whereas Pol II does. Transcription initiation by Pol I, which synthesizes pre-rRNA, and by Pol III, which synthesizes tRNAs, 5S rRNA, and other small stable RNAs (see Table 9-2), is tightly coupled to the rate of cell growth and proliferation.

Initiation by Pol I The regulatory elements directing Pol I initiation are similarly located relative to the transcription start site in yeast and in mammals. A core element spanning the transcription start site from −40 to +5 is essential for Pol I transcription. An additional upstream control element extending from roughly −155 to −60 increases in vitro Pol I transcription tenfold. In humans, assembly of the Pol I preinitiation complex (Figure 9-51) is initiated by the cooperative binding of UBF (upstream binding factor) and SL1 (selectivity factor), a multisubunit factor containing TBP and four Pol I–specific TBP-associated factors (TAFIs), to the Pol I promoter region. The TAFI subunits interact directly with Pol I–specific subunits, directing this specific nuclear RNA polymerase to the transcription start site. TIF-1A, the mammalian homolog of S. cerevisiae RRN3, is another required factor, as are the abundant nuclear protein kinase CK2 (casein kinase 2), nuclear actin, nuclear myosin, the protein deacetylase SIRT7, and topoisomerase I, which prevents DNA supercoils (see Figure 5-8) from forming during rapid Pol I transcription of the 14-kb transcription unit.

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FIGURE 9-51 Transcription of the rRNA precursor RNA by RNA polymerase I. (Top) Electron micrograph of RNA-protein complexes transcribed from one copy of the repeated rRNA genes. (Middle) A single Pol I transcription unit. Enhancers that stimulate Pol I transcription from a single transcription start site are represented by blue boxes. Pol I transcription termination sites (T0, T1–T10) bound by the Pol I–specific termination factor TTF-1 are shown as red rectangles. pRNA indicates transcription of the noncoding pRNA required for transcriptional silencing. The sequences of regions of DNA shown as yellow rectangles are retained during processing of 18S, 5.8S and 28S rRNAs. The other regions transcribed from the black arrow to the red termination sites are removed and degraded. (Bottom) The core promoter element and upstream control element are shown with the location of Pol I and its general transcription factors UBF, SL1, and TIF-1A represented, as well as other proteins required for Pol I elongation and control. See I. Grummt, 2010, FEBS J. 277:4626.
[Electron micrograph courtesy Ann L. Beyer.]

Transcription of the 14-kb precursor of 18S, 5.8S, and 28S rRNAs (see Chapter 10) is highly regulated to coordinate ribosome synthesis with cell growth and division. This coordination is achieved through regulation of the activities of the Pol I initiation factors by post-translational modifications, including phosphorylation and acetylation at specific sites, control of the rate of Pol I elongation, and control of the number of the 300 or so human rRNA genes that are transcriptionally active by epigenetic mechanisms that assemble inactive copies into heterochromatin. Switching between the active and heterochromatic silent states of rRNA genes is accomplished by a multisubunit chromatin-remodeling complex called NoRC (“No” for nucleolus, the site of rRNA transcription within nuclei). NoRC localizes a nucleosome over the Pol I transcription start site, blocking preinitiation complex assembly. It also interacts with a DNA methyl transferase that methylates a critical CpG in the upstream control element, inhibiting binding by UBF, as well as with histone methyl transferases that di- and trimethylate histone H3 lysine 9, creating binding sites for heterochromatic HP1, and with histone deacetylases. Moreover, a roughly 250-nt noncoding RNA called pRNA (promoter-associated RNA) transcribed by Pol I from about 2 kb upstream of the rRNA transcription unit (red arrow in Figure 9-51) is bound by a subunit of NoRC and is required for transcriptional silencing. The pRNA is believed to target NoRC to Pol I promoter regions by forming an RNA:DNA triplex with the T0 terminator sequence. This creates a binding site for the DNA methyl transferase DNMT3b, which methylates the critical CpG in the upstream promoter element.

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Initiation by Pol III Unlike those of protein-coding genes and pre-rRNA genes, the promoter regions of tRNA and 5S-rRNA genes lie entirely within the transcribed sequence (Figure 9-52a, b). Two such internal promoter elements, termed the A box and the B box, are present in all tRNA genes. These highly conserved sequences not only function as promoters, but also encode two invariant portions of eukaryotic tRNAs that are required for protein synthesis. In 5S-rRNA genes, a single internal control region, the C box, acts as a promoter.

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FIGURE 9-52 Transcription-control elements in genes transcribed by RNA polymerase III. Both tRNA (a) and 5S-rRNA (b) genes contain internal promoter elements (yellow) located downstream from the start site and named A, B, and C boxes, as indicated. Assembly of transcription initiation complexes on these genes begins with the binding of Pol III–specific general transcription factors TFIIIA, TFIIIB, and TFIIIC to these control elements. Green arrows indicate strong, sequence-specific protein-DNA interactions. Blue arrows indicate interactions between general transcription factors. Purple arrows indicate interactions between general transcription factors and Pol III. (c) Transcription of the U6 snRNA gene in mammals is controlled by an upstream promoter with a TATA box bound by the TBP subunit of a specialized form of TFIIIB with an alternative BRF subunit and an upstream regulatory element called the PSE bound by a multisubunit factor called SNAPC. See L. Schramm and N. Hernandez, 2002, Gene Dev. 16:2593.

Three general transcription factors are required for Pol III to initiate transcription of tRNA and 5S-rRNA genes in vitro. Two multimeric factors, TFIIIC and TFIIIB, participate in initiation at both tRNA and 5S-rRNA promoters; a third factor, TFIIIA, is required for initiation at 5S-rRNA promoters. As with assembly of Pol I and Pol II initiation complexes, the Pol III general transcription factors bind to promoter DNA in a defined sequence.

The N-terminal half of one TFIIIB subunit, called BRF (for TFIIB-related factor), is similar in sequence to TFIIB (a Pol II factor). This similarity suggests that BRF and TFIIB perform a similar function in initiation, namely, to assist in separating the template DNA strands at the transcription start site. Once TFIIIB has bound to either a tRNA or a 5S-rRNA gene, Pol III can bind and initiate transcription in the presence of ribonucleoside triphosphates. The BRF subunit of TFIIIB interacts specifically with one of the polymerase subunits unique to Pol III, accounting for initiation by this specific nuclear RNA polymerase.

Another of the three subunits composing TFIIIB is TBP, which we can now see is a component of a general transcription factor for all three eukaryotic nuclear RNA polymerases. The finding that TBP participates in transcription initiation by Pol I and Pol III was surprising, since the promoters recognized by these enzymes often do not contain TATA boxes. Nonetheless, in the case of Pol III transcription, the TBP subunit of TFIIIB interacts with DNA about 30 bp upstream of the transcription start site similarly to the way it interacts with TATA boxes.

Pol III also transcribes genes for small stable RNAs with upstream promoters containing a TATA box. One example is the gene for U6 snRNA, which is involved in pre-mRNA splicing, as discussed in Chapter 10. In mammals, this gene contains an upstream promoter element called the PSE in addition to the TATA box (Figure 9-52c). The PSE is bound by a multisubunit complex called SNAPC, while the TATA box is bound by the TBP subunit of a specialized form of TFIIIB containing an alternative BRF subunit.

MAF1 is a specific inhibitor of Pol III transcription that functions by interacting with the BRF subunit of TFIIIB and with Pol III. Its function is regulated by control of its import from the cytoplasm into the nucleus by phosphorylations at specific sites in response to signal transduction protein kinase cascades that respond to cell stress and nutrient deprivation (see Chapters 16 and 24). In mammals, Pol III transcription is also repressed by the critical tumor suppressors p53 and the retinoblastoma (Rb) family. In humans, there are two genes encoding RNA polymerase III subunit RPC32. One of these is expressed specifically in replicating cells, and its forced expression can contribute to oncogenic transformation of cultured human fibroblasts.

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