A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.

To directly map the position of promoter DNA within the RNA Polymerase II (Pol II) transcription Preinitiation Complex (PIC), FeBABE was tethered to specific sites within the HIS4 promoter and used to map exposed surfaces of Pol II and the general transcription factors in proximity to DNA. Our results distinguish between previously proposed models for PIC structure and demonstrate that downstream promoter DNA is positioned over the central cleft of Pol II with DNA upstream of TATA extending toward the Pol II subunit Rpb3. Also mapped were segments of TFIIB, IIE, IIF, and IIH in proximity to promoter DNA. DNA downstream of the transcription bubble maps to a path between the two helicase subdomains of the TFIIH subunit Rad25 (XPB). Together, our results show how the general factors and Pol II converge on promoter DNA within the PIC. KeywordsRNA Pol II; preinitiation complex; hydroxyl radical; general transcription factor Transcription of protein-coding genes by Pol II requires the recruitment of Pol II and the general transcription factors (GTFs) to promoters to form a PIC 1-3 , analogous to the Closed Complex state of bacterial RNAP. Prior to transcription initiation, the PIC transitions into the Open Complex state, where ATP and the TFIIH helicase subunit XPB promote melting of DNA surrounding the transcription start site and positioning of the template strand of the promoter within the active site cleft of Pol II 1,4-7 . In metazoans, ∼12 base pairs (bp) of DNA are melted in the Open Complex, and transcription typically initiates at a single start site ∼30-bp downstream from the TATA element. However, transcription start site selection in S. cerevisiae is more complex since initiation occurs between 40−120-bp from TATA 8 . Despite these differences in start site position, the upstream edge of the transcription bubble maps to ∼20-bp downstream of TATA both in metazoan systems and S. cerevisiae 7,9-11 . These observations led to the suggestion that yeast Pol II "scans" downstream DNA for a suitable initiation site 9 .Much insight into the mechanism of Pol II transcription has been gained from crystal structures of yeast . Structural studies have shown that for elongating Pol II, the template DNA strand lies inside the deep central cleft located between Rpb1 and Rpb2, the two largest Email: shahn@fhcrc.org, Telephone: 206 667 5261, Fax: 206 667 6497. Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating PubMed Central (PMC) documents. PMC documents will be made available for review after conversion (approx. 2−3 weeks time). Any corrections that need to be made will be done at that time. No materials will be released to PMC without the approval of an author. Only the PMC documents will appear on PubMed Central --this PDF Receipt will not appear on PubMed Central. More recently, separate studies examining TFIIB interaction with Pol II have provided two additional models of PIC structure 23-25 . X-ray structure analysis of a complex of Pol II and TFIIB 23 showed that the TFI...