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Eukaryotic transcription factors: Class I and class III. Class I promoters •Service rRNA precursor gene •Sequences are variable from one species to another •Two critical regions: core element, at the start of transcription, between -45 and +20 upstream promoter element (UPE), between positions –156 and –107. 1 Class I promoter elements Linker scanning was used to identify important regions, tested using in vitro transcription Class I factors Much simpler than class II Preinitiation complex is much simpler: •Polymerase I •Two transcription factors: ~ SL1 in humans and TIF-IB in other organisms – core binding factors ~ upstream-binding factor, UBF (mammals) or upstream activating factor, UAF (yeast) – assembly factors 2 SL1 factor Discovered in 1985 from HeLa cells. Is a species-specific factor. Species-specificity of SL1 TF: it could distinguish mouse and human rRNA template. Necessary and sufficient to recruit polymerase. SL1 also determines species specificity, as does the core promoter element. UPE-binding factor UBF •UBF – a transcription factor that stimulates transcription by polymerase I; composed of two polypeptides, 94 and 97 kD •97 kD alone is sufficient for UBF activity •UBF and SL1 act synergistically to stimulate transcription. 3 Activation of transcription from rRNA promoter by SL1 and UBF S1 assay was used to measure transcription from human rRNA promoter in the presence of RNA polymerase I and various combinations of UBF and SL1. Conclusion: SL1 was required for at least basal activity, but UBF enhanced this activity. They act synergistically. Structure and function of SL1 TBP is essential for class I transcription. How? Tjian et al, 1992: SL1 is composed of TBP and 3 TAFs These are: TAFI110, TAFI63, TAFI48. Fully functional and species-specific SL1 can be reconstituted from those components. Binding of TBP to TAFIs precludes binding of it to TAFIIs. 4 Class III factors 1980, Roeder et al discovered TFIIIA, that was bound to 5S rRNA gene. Roeder et al discovered that TFIIIA was needed for 5S rRNA gene transcription, but not for the tRNA gene transcription. 1982 - Other factors: TFIIIB, TFIIIC were separated, they are necessary for the III class genes. 5SrRNA – needs TFIIIA+TFIIIB+TFIIIC Class III promoters RNA polymerase III genes: Classical – 5SrRNA, AV RNA Non-classical (recently discovered) – U6 snRNA, EpsteinBarr virus EBER2 gene – resemble class II promoters. Feature of class III – located within the genes themselves! 5 ‘Classical’ Class III genes with internal promoters Findings of Brown et al, works with X. laevis 5SrRNA TFIIIA Binds DNA, has zinc-finger domain – finger shaped domain that has aa that bind single Zn ion. In TFIIIA the 4 aa are cysteines, 2 are histidines. Has 9 Zn-fingers, it inserts them into the major groove at the region of promoter – tight DNA-protein complex is formed. 6 TFIIIB and TFIIIC Classical class III factors require TFIIIB and TFIIIC. Transcription of 5S rRNA gene depends upon TFIIIA, TFIIIB and TFIIIC: TFIIIC and A bind to internal promoter and help TFIIIB to bind to the region upstream the transcription start. TFIIIB remains bound and helps with the next rounds of transcription. Role of TBP in the class III transcription TFIIIC plays ‘organizing’ role as TBP to TATA-containing pol II genes ?TBP is required for tRNA and 5S rRNA genes? •TFIIIC binds to internal promoter’s A and B blocks. •Promotes binding of TFIIIB, with its TPB to the region upstream of transcription start site. •TFIIIB promotes binding of polymerase at the start site. •Transcription begins. When polymerase moves forward making RNA, it possibly removes TFIIIC from the promoter. •TFIIIB remains in place, ready to assist in anew round of polymerase binding and transcription. 7 Role of TBP in the class III transcription Model of preinitiation complex formation on TATA-less promoters recognized by all three types of polymerases 1) Assembly factor binds first (UBP, Sp1 and TFIIIC in class I, II and III promoters, respectively) 2) This attracts another factor that contains TBP (these are SL1, TFIID or TFIIIB in class I, II and III promoters, respectively). 3) These complexes are sufficient to recruit polymerase in class I and III 4) In class II more GTFs are required. 8 Model of preinitiation complex formation on TATA-less promoters recognized by all three types of polymerases 1) Assembly factor binds first (UBP, Sp1 and TFIIIC in class I, II and III promoters, respectively) 2) This attracts another factor that contains TBP (these are SL1, TFIID or TFIIIB in class I, II and III promoters, respectively). 3) These complexes are sufficient to recruit polymerase in class I and III 4) In class II more GTFs are required. The assembly of preinitiation complex begins with attachment of an assembly factor to promoter. TBP plays important role in assembly. Even if it is not the first bound factor, it becomes a part of growing complex. The specificity of TBP –which kind of promoter it binds depends upon TAFs. 9 Reading: Class I and class III factors, pages 327-339. 10