Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II.

A protocol for the incorporation of SeMet into yeast proteins is described. Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II. This shows the utility of the approach as an aid in the model-building of large protein complexes.

Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Yeast RNA polymerase II at 5 A resolution.

Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.

Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7.

The three-dimensional structure of wild-type yeast RNA polymerase II has been determined at a nominal resolution of 24 A. A difference map between this structure and that of the polymerase lacking subunits Rpb4 and Rpb7 showed these two subunits forming part of the floor of the DNA-binding (active center) cleft, and revealed a slight inward movement of the protein domain surrounding the cleft. Surface plasmon resonance measurements showed that Rpb4 and Rpb7 stabilize a minimal pre-initiation complex containing promoter DNA, TATA box-binding protein (TBP), transcription factor TFIIB and the polymerase. These findings suggest that Rpb4 and Rpb7 play a role in coupling the entry of DNA into the active center cleft to closure of the cleft. Such a role can explain why these subunits are necessary for promoter-specific transcription in vitro and for a normal stress response in vivo.

The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain.

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.

The UL8 subunit of the heterotrimeric herpes simplex virus type 1 helicase-primase is required for the unwinding of single strand DNA-binding protein (ICP8)-coated DNA substrates.

The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8. The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.

Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits.

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

Yeast RNA polymerase II transcription reconstituted with purified proteins.

Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation. This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

A yeast transcriptional stimulatory protein similar to human PC4.

A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator. Phosphorylation appears to regulate these interactions. The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability. The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4.

A minimal set of RNA polymerase II transcription protein interactions.

All pairwise interactions of RNA polymerase II and general transcription factors (TF) IIB, E, F, and H have been quantitated by surface plasmon resonance with the use of a Ni2+ chelate on the sensor surface where necessary to attain higher sensitivity. Only 4 of 10 possible interactions were found above the detection limit: TFIIB, -E, and -F binding to RNA polymerase II and TFIIE binding to TFIIH. These four interactions constitute a minimal set for the formation of a transcription initiation complex and may represent the primary interactions involved in assembly of the complex. Point mutations in TFIIB that alter the location of transcription start sites in vivo markedly diminished the affinity of TFIIB binding to RNA polymerase II. Protein blotting revealed an interaction between the largest subunit of TFIIE and third largest subunit of TFIIH, which may be responsible for TFIIE binding to TFIIH.

The C-terminal domain revealed in the structure of RNA polymerase II.

The location of the CTD in the structure of RNA polymerase II has been determined by electron crystallography at 16 A resolution. Difference maps between wild-type enzyme and that lacking the CTD, or with an antibody fragment bound in place of the CTD, disclose the site of attachment of the CTD to the polymerase. Appropriate display of the polymerase structure reveals the CTD as an element projecting from this site of attachment into solution. A low relative density and large volume of this element identify the CTD as a conformationally mobile region.

Two-dimensional crystallography of TFIIB- and IIE-RNA polymerase II complexes: implications for start site selection and initiation complex formation.

SUMMARY: Transcription factors IIB (TFIIB) and IIE (TFIIE) bound to RNA polymerase II have been revealed by electron crystallography in projection at 15.7 A resolution. The results lead to simple hypotheses for the roles of these factors in the initiation of transcription. TFIIB is suggested to define the distance from TATA box to transcription start site by bringing TATA DNA in contact with polymerase at that distance from the active center of the enzyme. TFIIE is suggested to participate in a key conformational switch occurring at the active center upon polymerase-DNA interaction.

Different forms of TFIIH for transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome.

Yeast TFIIH that is active in transcription can be dissociated into three components: a 5-subunit core, the SSL2 gene product, and a complex of 47 kDa, 45 kDa, and 33 kDa polypeptides that possesses protein kinase activity directed towards the C-terminal repeat domain of RNA polymerase II. These three components can reconstitute fully functional TFIIH, and all three are required for transcription in vitro. By contrast, TFIIH that is highly active in nucleotide excision repair (NER) lacks the kinase complex and instead contains the products of all other genes known to be required for NER in yeast: RAD1, RAD2, RAD4, RAD10, and RAD14. This repairosome is not active in reconstituted transcription in vitro and is significantly more active than any of the constituent polypeptides in correcting defective repair in extracts from strains mutated in NER genes.

Yeast TFIIE. Cloning, expression, and homology to vertebrate proteins.

Genes encoding both the 66- and the 43-kDa subunits of yeast RNA polymerase II initiation factor a, designated TFA1 and TFA2, have been isolated. Both genes are essential for cell viability. The bacterially expressed gene products could replace factor a in transcription in vitro, and both recombinant subunits were required for activity. The deduced amino acid sequences of the TFA1 and TFA2 gene products were homologous to those of the large and small subunits of human TFIIE, respectively, identifying factor a as the yeast homolog of TFIIE.