Preinitiation complex

The DNA part of each control module can be divided into three main regions, the core or basal promoter elements, the promoter proximal elements and the distal enhancer elements (Figure 9.1). The best characterized core promoter element is the TATA box, a DNA sequence that is rich in A-T base pairs and located 25 base pairs upstream of the transcription start site. The TATA box is recognized by one of the basal transcription factors, the TATA box-binding protein, TBP, which is part of a multisubunit complex called TFIID. This complex in combination with RNA polymerase 11 and other basal transcription factors such as TFIIA and TFIIB form a preinitiation complex for transcription. 

The promoter proximal elements are usually 100 to 200 base pairs long and relatively close to the site of initiation of transcription. Within each of these elements there are DNA sequences specifically recognized by several different transcription factors which either interact directly with the preinitiation complex or indirectly through other proteins. 

Figure 9.2 Schematic model for transcriptional activation. The TATA box-binding protein, which bends the DNA upon binding to the TATA box, binds to RNA polymerase and a number of associated proteins to form the preinitiation complex. This complex interacts with different specific transcription factors that bind to promoter proximal elements and enhancer elements.

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

TFIIA and TFIIB are two basal transcription factors that are involved in the nucleation stages of the preinitiation complex by binding to the TBP-TATA box complex. Crystal structures of the ternary complex TFIIA-TBP-TATA box have been determined by the groups of Paul Sigler, Yale University, and Timothy Richmond, ETH, Zurich, and that of the TFIIB-TBP-TATA box by Stephen Burley and collaborators. The TBP-DNA interactions and the distortions of the DNA structure are essentially the same in these ternary complexes as in the binary TBP-TATA complex. 

DNA binding by TBP is strongly dependent on the presence of T-A base pairs in the TATA box. Bending allows remote sites on the DNA, with their bound cognate specific transcription factors, to come close together such that the proteins can interact to form the transcriptional preinitiation complex. 

Template binding RNA polymerase (RNAP) binds to DNA and locates a promoter (P) melts the two DNA strands to form a preinitiation complex (PIQ. (2) Chain initiation RNAP holoenzyme (core + one of multiple sigma factors) catalyzes the coupling of the first base (usually ATP or GTP) to a second ribonucleoside triphosphate to form a dinucleotide. (3) Chain elongation Successive residues are added to the 3 -OH terminus of the nascent RNA molecule. (4) Chain termination and release The completed RNA chain and RNAP are released from the template. The RNAP holoenzyme re-forms, finds a promoter, and the cycle is repeated. 

Figure 37-7. Transcription elements and binding factors in the herpes simplex virus thymidine kinase ffW gene. DNA-dependent RNA polymerase II binds to the region of the TATA box (which is bound by transcription factor TEND) to form a multicomponent preinitiation complex capable of initiating transcription at a single nucleotide (+1).The frequency of this event is increased by the presence of upstream c/s-acting elements (the GC and CAAT boxes). These elements bind frans-acting transcription factors, in this example Spl and CTF (also called C/EBP, NF1, NFY). These cis elements can function independently of orientation (arrows).

RNA polymerases interact with unique cw-active regions of genes, termed promoters, in order to form preinitiation complexes (PICs) capable of initiation. In eukaryotes the process of PIC formation is facilitated by multiple general transcription factors (GTFs), TFIIA, B, D, E, F, and H. 

Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome. Once the mRNA binds to the ribosome, the latter finds the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (elFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps (1) dissociation of the ribosome into its 40S and 60S subunits (2) binding of a ternary complex consisting of met-tRNAf GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex (3) binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex and (4) combination of the 43S initiation complex with the 60S ribosomal subunit to form the SOS initiation complex. 

The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met-tRNAf a tRNA specifically involved in binding to the initiation codon AUG. (There are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence.) This ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and elF-lA. 

Figure 7.5 Model of ferritin (and erythroid a-aminolaevulinate synthase) translation/ribosome binding regulation by IRP. In (a), with IRP not bound to the IRE (1) binding of the 43S preinitiation complex (consisting of the small ribosomal 40S subunit, GTP and Met-tRNAMet) to the mRNA is assisted by initiation factors associated with this complex, as well as additional eukaryotic initiation factors (elFs) that interact with the mRNA to facilitate 43S association. Subsequently (2), the 43S preinitiation complex moves along the 5 -UTR towards the AUG initiator codon, (3) GTP is hydrolysed, initiation factors are released and assembly of the 80S ribosome occurs. Protein synthesis from the open reading frame (ORF) can now proceed. In (b) With IRP bound to the IRE, access of the 43S preinitiation complex to the mRNA is sterically blocked. From Gray and Hentze, 1994, by permission of Oxford University Press.

Singh, C. R., Curtis, C., Yamamoto, Y., Hall, N. S., Kruse, D. S., He, H., Hannig, E. M., and Asano, K. (2005). Eukaryotic translation initiation factor 5 is critical for integrity of the scanning preinitiation complex and accurate control of GCN4 translation. Mol. Cell. Biol. 25, 5480-5491. 

Valasek, L., Nielsen, K. H., Zhang, F., Fekete, C. A., and Hinnebusch, A. G. (2004). Interactions of Eukaryotic Translation Initiation Factor 3 (eIF3) Subunit NIPl/c with elFl and eIF5 promote preinitiation complex assembly and regulate start codon selection. Mol. Cell. Biol. 24, 9437-9455. 

Kedersha, N., Chen, S., Gilks, N., Li, W., Miller, I. J., Stahl, J., and Anderson, P. (2002). Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell 13, 195—210. 

Pestova, T. V., Shatsky, I. N., and Hellen, C. U. (1996b). Functional dissection of eukaryotic initiation factor 4F The 4 A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol. Cell Biol. 16, 6870-6878. 

These may enhance or obstruct the assembly of the preinitiation complex of RNA polymerase and relevant transcription factors [9]. Physiological isoform expression varies with cell type and maturity. In rat heart, the predominant form shifts from neonatal a3 to adult a2. 

CREB-mediated transcription are evident at the level of preinitiation complex recruitment. These data suggest a complexity of CREB-responsive gene expression that probably involves several co-regulators beyond the simple acetyltransferase mediators. 

Lu W, Peterson R, Dasgupta A, Scovell WM (2000) Influence of HMG-1 and adenovirus oncoprotein ElA on early stages of transcriptional preinitiation complex assembly. J Biol Chem 275 35006-35012 Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucle-osome core particle at 2.8 A resolution. Nature 389 251—260 Lusser A, Kadonaga JT (2004) Strategies for the reconstitution of chromatin. Nat Methods 1 19-26 Maeshima K, Laemmli UK (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev Cell 4 467-480... 

Jiang W, Nordeen SK, Kadonaga JT (2000) Transcriptional analysis of chromatin assembled with purified ACF and dNAPl reveals that acetyl-CoA is required for preinitiation complex assembly. J.Biol.Chem 22 39819-39822... 

Nogales E (2000) Recent structural insights into transcription preinitiation complexes. J Cell Sci 113 Pt... 

In vitro interactions between HMG proteins and the basal transcription machinery have also been reported. Human HMGBl binds to the TATA-box binding protein (TBP) and interferes with the normal binding of TFIIB in the preinitiation complex [154,155], thereby inhibiting TBP function both HMGBl and TFIIB independently enhance binding of TBP to TATA-box DNA [154]. Similarly, Nhp6ap promotes the formation of a complex with TBP and TFIIA at the TATA... 

Pauli, T.T., Carey, M., and Johnson, R.C. (1996) Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro. Genes Dev. 10, 2769-2781. 

The potential impact of the chromatin structure on ERa- and ER/1-mediated transcriptional activities was investigated using an in vitro chromatin assembly assay. These experiments have shown that the AF-1 domain of ERa, but not of ER/1, contains a transferable activation domain, which permits the ERa to efficiently activate transcription on chromatin templates [58]. Furthermore, the co-activators CBP/p300 and SRC have to be recruited to the ERa in order to maximally enhance transcription on ERa-susceptible chromatin templates. The p300/CBP-SRC complex, when interacting with the AF-1 of the ERa, is primarily involved in the stable formation of the preinitiation complex of transcription [59]. 

Kosa, P. E, Ghosh, G., DeDecker, B. S. and Sigler, P. B. (1997). The 2.1-A crystal structure of an archaeal preinitiation complex TATA-box-binding protein/transcription factor (II)B core/TATA-box. Proc. Nat. Acad. Sci. USA 94, 6042-6047. 

Tsai, E T.E. and Sigler, P. B. (2000). Structural basis of preinitiation complex assembly on human Pol 11 promoters. EMBO. 19,25-36. 

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