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 The 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 Initiation is followed by promoter clearance A connection between transcription and repair Promoters for RNA polymerase II have short sequence elements Some promoter-binding proteins are repressors Enhancers contain bidirectional elements that assist initiation Independent domains bind DNA and activate transcription The two hybrid assay detects protein-protein interactions Interaction of upstream 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 Introduction

Figure 20.1 A typical gene transcribed 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 may 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 transcription by certain eukaryotic RNA polymerases, especially RNA polymerase II Eukaryotic RNA polymerases consist of many subunits

Figure 20.2 Eukaryotic RNA polymerase II has >10 subunits Eukaryotic RNA polymerases consist of many subunits

Cotransfection is the simultaneous transfection of two markers 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 the promoter must lie between them 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 then 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 RNA polymerase I has a bipartite promoter

Preinitiation complex in eukaryotic transcription describes the assembly of transcription factors at the promoter before RNA polymerase binds 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 RNA polymerase III uses 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) RNA polymerase 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 RNA polymerase III uses both downstream and upstream promoters

TATA box is a conserved A · T-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 The startpoint for RNA polymerase II

Figure 20.8 RNA polymerases are positioned at all promoters by a factor that contains TBP 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 is shown in green. The two strands of the DNA double helix are in light and dark grey. Photograph kindly provided by Stephen Burley TBP is a universal factor

Figure 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 TBP is a universal factor

Figure An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of association with transcription factors The basal apparatus assembles at the promoter

Figure Two views of the ternary complex of TFIIB-TBP-DNA show that TFIIB binds along the bent face of DNA. The two strands of DNA are green and yellow, TBP is blue, and TFIIB is red and purple. Photograph kindly provided by Stephen Burley The basal apparatus assembles at the promoter

Figure Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription The basal apparatus assembles at the promoter

Figure Mfd recognizes a stalled RNA polymerase and directs DNA repair to the damaged template strand A connection between transcription and repair

Figure The Uvr system operates in stages in which UvrAB recognizes damage, UvrBC nicks the DNA, and UvrD unwinds the marked region A connection between transcription and repair

Figure The TFIIH core may associate with a kinase at initiation and associate with a repair complex when damaged DNA is encountered A connection between transcription and repair

Figure A helicase unwinds DNA at a damaged site, endonucleases cut on either side of the lesion, and new DNA is synthesized to replace the excised stretch A connection between transcription and repair

CAAT box is part of a conserved sequence located upstream of the startpoints of eukaryotic transcription units; it is recognized by a large group of transcription factors Promoters for RNA polymerase II have short sequence elements

Figure Saturation mutagenesis of the upstream region of the  - globin promoter identifies three short regions (centered at -30, - 75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, Promoters for RNA polymerase II have short sequence elements

Figure Promoters contain different combinations of TATA boxes, CAAT boxes, GC boxes, and other elements Promoters for RNA polymerase II have short sequence elements

Table Upstream transcription factors bind to sequence elements that are common to mammalian RNA polymerase II promoters Promoters for RNA polymerase II have short sequence elements ModuleConsnesus DNA bound Factor TATA box TATAAAA~10bpTBP CAAT box GGCCAATCT~22bpCTF/NF1 GC box GGGCGG~20bpSP1 OctamerATTTGCAT~20bpOct-1 OctamerATTTGCAT~23bpOct-2 kBGGGACTTTCC~10bp NF kB ATFGTGACGT~20bpATF

Table Upstream transcription factors bind to sequence elements that are common to mammalian RNA polymerase II promoters Promoters for RNA polymerase II have short sequence elements ModuleConsnesus DNA bound Factor TATA box TATAAAA~10bpTBP CAAT box GGCCAATCT~22bpCTF/NF1 GC box GGGCGG~20bpSP1 OctamerATTTGCAT~20bpOct-1 OctamerATTTGCAT~23bpOct-2 kBGGGACTTTCC~10bp NF kB ATFGTGACGT~20bpATF

Figure A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form Promoters for RNA polymerase II have short sequence elements

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 Enhancers contain bidirectional elements that assist initiation

Figure Indirect end- labeling identifies the distance of a DNAase hypersensitive site from a restriction cleavage site. The existence of a particular cutting site for DNAase I generates a discrete fragment, whose size indicates the distance of the DNAase I hypersensitive site from the restriction site Enhancers contain bidirectional elements that assist initiation

Figure The SV40 minichromosome has a nucleosome gap. Photograph kindly provided by Moshe Yaniv Enhancers contain bidirectional elements that assist initiation

Figure An enhancer contains several structural motifs. The histogram plots the effect of all mutations that reduce enhancer function to <75% of wild type. Binding sites for proteins are indicated below the histogram Enhancers contain bidirectional elements that assist initiation

Figure Saturation mutagenesis of the upstream region of the  -globin promoter identifies three short regions (centered at -30, -75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, Enhancers contain bidirectional elements that assist initiation

Figure An enhancer may function by bringing proteins into the vicinity of the promoter. An enhancer does not act on a promoter at the opposite end of a long linear DNA, but becomes effective when the DNA is joined into a circle by a protein bridge. An enhancer and promoter on separate circular DNAs do not interact, but can interact when the two molecules are catenated Enhancers contain bidirectional elements that assist initiation

Figure DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein Independent domains bind DNA and activate transcription

Figure The GAL4 protein has independent regions that bind DNA, activate transcription (2 regions), dimerize, and bind the regulator GAL Independent domains bind DNA and activate transcription

Figure The ability of GAL4 to activate transcription is independent of its specificity for binding DNA. When the GAL4 DNA-binding domain is replaced by the LexA DNA-binding domain, the hybrid protein can activate transcription when a LexA operator is placed near a promoter Independent domains bind DNA and activate transcription

Figure The activating domain of the tat protein of HIV can stimulate initiation if it is tethered in the vicinity by binding to the RNA product of a previous round of transcription. Activation is independent of the means Independent domains bind DNA and activate transcription

Figure The two hybrid technique tests the ability of two proteins to interact by incorporating them into hybrid proteins where one has a DNA-binding domain and the other has a transcription- activating domain Independent domains bind DNA and activate transcription

Figure DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein Interaction of upstream factors with the basal apparatus

Figure An upstream transcription factor may bind a coactivator that contacts the basal apparatus Interaction of upstream factors with the basal apparatus

Figure The activating domain of the tat protein of HIV can stimulate initiation if it is tethered in the vicinity by binding to the RNA product of a previous round of transcription. Activation is independent of the means Interaction of upstream factors with the basal apparatus

Figure An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of association with transcription factors Interaction of upstream factors with the basal apparatus

Figure Upstream activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB Interaction of upstream factors with the basal apparatus

1. Of the three eukaryotic RNA polymerases, RNA polymerase I transcribes rDNA and accounts for the majority of activity, RNA polymerase II transcribes structural genes for mRNA and has the greatest diversity of products, and RNA polymerase III transcribes small RNAs. 2. None of the three RNA polymerases recognize their promoters directly. 3. The TATA box (if there is one) near the startpoint, and the initiator region immediately at the startpoint, are responsible for selection of the exact startpoint at promoters for RNA polymerase II. 4. RNA polymerase is found as part of much larger complexes that contain factors that interact with activators and repressors. 5. Promoters for RNA polymerase II contain a variety of short cis-acting elements, each of which is recognized by a trans-acting factor. 6. Promoters may be stimulated by enhancers, sequences that can act at great distances and in either orientation on either side of a gene. Summary