Biochemistry 201RNA polymerase II transcription apparatusProfessor Roger KornbergFebruary 7, 2000RNA polymerase II of eukaryotes transcribes all but a few genes in eukaryotes,RNA polymerase I transcribes the genes for the two large ribosomal RNAs,and RNA polymerase III transcribes genes for tRNAs. RNA polymerase IIfrom the yeast Saccharomyces cerevisiae has been studied in most detail. It iscomposed of 15 polypeptides (denoted Rpb1-12, with two copies each of Rpb3,Rpb5 and Rpb9), with a total molecular mass of 587 kDa. The two largestsubunits, Rpb1 and Rpb2, exhibit extensive sequence conservation with β andβ' of E. coli, in 8 and 9 regions of homology, respectively. These homologyregions are conserved in the human proteins as well.Rpb1 contains an additional, unusual feature, a repeated heptapeptide at its3'-terminus. There are 26 repeats in yeast, 45 in Drosophila, and 52 in man.The consensus heptapeptide sequence is identically conserved from yeast toman. The CTD undergoes a cycle of extensive phosphorylation anddephosphorylation accompanying every round of transcription. Theunphosphorylated form, designated IIa, initiates transcription, while thehyperphosphorylated form, designated IIo, is associated with elongationcomplexes.RNA polymerase II is alone incapable of recognizing a promoter and intiatingtranscription. It requires a set of additional proteins termed generaltranscription factors. Five such factors, termed TFIIB, -D, -E, -F, and -H arerequired for transcription of all promoters by purified RNA polymerase II in acell-free system. TFIID can be replaced by one of its subunits, TATA-bindingprotein (TBP), for transcription of a core or minimal promoter, consisting ofonly a TATA box and transcription start site. The distance from TATA box totranscription start site is about 25 base pairs in almost all polymerase IIpromoters in almost all organisms. This conserved spacing is the hallmark ofa polymerase II promoter.Transcription factors B, E, and F interact directly with RNA polymerase II. Bcouples to TBP and the TATA box, while E couples to H. The largest subunitof H is an ATPase/helicase, which unwinds double stranded DNA around thestart site as required for transcription. Other subunits of H supply the kinaseactivity responsible for CTD phosphorylation accompanying the initiation oftranscription. Both helicase and kinase subunits of H relate to other majorcellular transactions. The helicase and five additional H subunits form partof a DNA repairosome, which includes other proteins required for nucleotideexcision DNA repair. This connection may account for the preferential repairof DNA damage in the transcribed strand in vivo. The H kinase belongs tothe family of cell cycle control protein kinases and includes a cyclin subunit aswell.Structural studies provide a rationale for the general transcription factors andtheir roles in the intiation mechanism. The TATA box is recognized byTATA-binding protein, whose exceptionally conserved C-terminal 180 aminoacid domain is sufficient for transcription. X-ray crystal structures have beendetermined for TBP alone, in a complex with TATA DNA, and in a ternarycomplex with TATA DNA and a large fragment of TFIIB. TBP is saddle-shaped, with a pseudo two-fold axis of symmetry relating two halves of the C-terminal domain which are similar but not identical in sequence. DNA bindsto the concave underside of the saddle, at an angle to the pseudo two-foldaxis. The DNA is partially unwound, from a helix to a "ladder", and TBP liesin the flat minor groove of the ladder. Kinks at the edges where DNA entersand leaves the TBP binding site result in a strongly bent shape of the boundDNA. TFIIB interacts with the DNA on both sides of TBP, whose role is, ineffect, to bend DNA to enable TFIIB binding.Electron and X-ray crystallography of cocrystals of yeast RNA polymerase IIwith transcription factors B and E identifies their sites of interaction on thesurface of the enzyme, leading to a simple hypothesis for the mechanism ofstart site determination and the intiation of transcription. B brings the TATAbox of a promoter to a point on the polymerase surface about 110Å from theactive center, corresponding to about 30 base pairs length of DNA, and therebyaccounting for the conserved spacing of TATA box and start site oftranscription. Entry of DNA triggers closure of the polymerase arm aroundthe active center cleft which, in turn, creates the site for E binding. E recruitsH, which melts the promoter to initiate transcription.X-ray crystallography has now revealed the structure of RNA polymerase II atnear atomic resolution. Electron crystallography of an actively transcribingcomplex identifies the site of interaction with duplex DNA in the structure.The combined results explain the remarkable aspects of transcription: thetight coupling between the presence of an RNA transcript and the grip of theenzyme on the DNA template, and the oscillation between forward andretrograde movement on the template.References:Buratowski, S., Hahn, S., Guarente, L., and Sharp, P.A. (1989) Fiveintermediate complexes in transcription initiation by RNA polymease II. Cell56:549-562.Bushnell, D. A., Bamdad, C., and Kornberg, R. D. (1996) A minimal set ofRNA polymerase II transcription protein interactions. J. Biol. Chem.271:20170-10174.Conaway, R.C. and Conaway, J.W. (1993) General initiation factors for RNApolymerase II. Annu. Rev. Biochem. 62:161-190.Darst, S. A., Edwards, A. M., Kubalek, E. W., and Kornberg, R. D. (1991) Three-dimensional structure of yeast RNA polymerase II at 16Å resolution. Cell66:121-128.Darst, S. A., Kubalek, E. W., and Kornberg, R. D. (1989) Three-dimensionalstructure of Escherichia coli RNA polymerase holoenzyme determined byelectron crystallography. Nature 340:730-732.Feaver, W. J., Gileadi, O., Li, Y., and Kornberg, R. D. (1991). CTD kinaseassociated with yeast RNA polymerase II initiation factor b. Cell 67:1223-1330.Feaver, W. J., Svejstrup, J. Q., Bardwell, L., Bardwell, A. J., Buratowski, S.,Gulyas, K. D., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1993). Dualroles of a multiprotein complex from S. cerevisiae in transcription and DNArepair. Cell 75:1379-1387.Feaver, W. J., Svejstrup, J. Q., Henry, N. L., and Kornberg, R. D.