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1、Chapter 20,Initiation of transcription,20.1 Introduction 20.2 Eukaryotic RNA polymerases consist of many subunits 20.3 Promoter elements are defined by mutations and footprinting 20.4 RNA polymerase I has a bipartite promoter 20.5 RNA polymerase III uses both downstream and upstream promoters 20.6 T
2、he startpoint for RNA polymerase II 20.7 TBP is a universal factor 20.8 TBP binds DNA in an unusual way 20.9 The basal apparatus assembles at the promoter 20.10 Initiation is followed by promoter clearance 20.11 A connection between transcription and repair 20.12 Promoters for RNA polymerase II have
3、 short sequence elements 20.13 Some promoter-binding proteins are repressors 20.14 Enhancers contain bidirectional elements that assist initiation 20.15 Independent domains bind DNA and activate transcription 20.16 The two hybrid assay detects protein-protein interactions 20.17 Interaction of upstre
4、am factors with the basal apparatus,Enhancer element is a cis-acting sequence that increases the utilization of (some) eukaryotic promoters, and can function in either orientation and in any location (upstream or downstream) relative to the promoter.,20.1 Introduction,Figure 20.1 A typical gene tran
5、scribed by RNA polymerase II has a promoter that extends upstream from the site where transcription is initiated. The promoter contains several short (200 bp. An enhancer containing a more closely packed array of elements that also bind transcription factors may be located several kb distant. (DNA m
6、ay be coiled or otherwise rearranged so that transcription factors at the promoter and at the enhancer interact to form a large protein complex.),20.1 Introduction,Amanitin (more fully a-amanitin)is a bicyclic octapeptide derived from the poisonous mushroom Amanita phalloides; it inhibits transcript
7、ion by certain eukaryotic RNA polymerases, especially RNA polymerase II.,20.2 Eukaryotic RNA polymerases consist of many subunits,Figure 20.2 Eukaryotic RNA polymerase II has 10 subunits.,20.2 Eukaryotic RNA polymerases consist of many subunits,Cotransfection is the simultaneous transfection of two
8、markers.,20.3 Promoter elements are defined by mutations and footprinting,Figure 20.3 Promoter boundaries can be determined by making deletions that progressively remove more material from one side. When one deletion fails to prevent RNA synthesis but the next stops transcription, the boundary of th
9、e promoter must lie between them.,20.3 Promoter elements are defined by mutations and footprinting,Figure 20.4 Transcription units for RNA polymerase I have a core promoter separated by 70 bp from the upstream control element. UBF1 binds to both regions, after which SL1 can bind. RNA polymerase I th
10、en binds to the core promoter. The nature of the interaction between the factors bound at the upstream control element and those at the core promoter is not known.,20.4 RNA polymerase I has a bipartite promoter,Preinitiation complex in eukaryotic transcription describes the assembly of transcription
11、 factors at the promoter before RNA polymerase binds.,20.5 RNA polymerase III uses both downstream and upstream promoters,Figure 20.5 Deletion analysis shows that the promoter for 5S RNA genes is internal; initiation occurs a fixed distance (55 bp) upstream of the promoter.,20.5 RNA polymerase III u
12、ses both downstream and upstream promoters,Figure 20.6 Promoters for RNA polymerase III may consist of bipartite sequences downstream of the startpoint, with boxA separated from either boxC or boxB. Or they may consist of separated sequences upstream of the startpoint (Oct, PSE, TATA).,20.5 RNA poly
13、merase III uses both downstream and upstream promoters,Figure 20.7 Initiation via the internal pol III promoters involves the assembly factors TFIIIA and TFIIIC, the initiation factor TFIIIB, and RNA polymerase III.,20.5 RNA polymerase III uses both downstream and upstream promoters,TATA box is a co
14、nserved AT-rich septamer found about 25 bp before the startpoint of each eukaryotic RNA polymerase II transcription unit; may be involved in positioning the enzyme for correct initiation.,20.6 The startpoint for RNA polymerase II,Figure 20.8 RNA polymerases are positioned at all promoters by a facto
15、r that contains TBP.,20.7 TBP is a universal factor,Figure 20.9 A view in cross-section shows that TBP surrounds DNA from the side of the narrow groove. TBP consists of two related (40% identical) conserved domains, which are shown in light and dark blue. The N-terminal region varies extensively and
16、 is shown in green. The two strands of the DNA double helix are in light and dark grey. Photograph kindly provided by Stephen Burley.,20.7 TBP is a universal factor,Figure 20.10 The cocrystal structure of TBP with DNA from -40 to the startpoint shows a bend at the TATA box that widens the narrow groove where TBP binds. Photograph provided by Stephen Burley.,20.7 TBP is a universal factor,Figure 20.11 An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of
