Positive regulation of general transcription factor SIII by a tailed ubiquitin homolog.

General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.

Molecular cloning of an essential subunit of RNA polymerase II elongation factor SIII.

A transcription factor designated SIII was recently purified from mammalian cells and shown to regulate the activity of the RNA polymerase II elongation complex. SIII is a heterotrimer composed of approximately 110-, 18-, and 15-kDa polypeptides and is capable of increasing the overall rate of RNA chain elongation by RNA polymerase II by suppressing transient pausing of polymerase at multiple sites on the DNA template. Here we describe the molecular cloning and characterization of a cDNA encoding the functional 15-kDa subunit (p15) of SIII. The p15 cDNA encodes a 112-amino-acid polypeptide with a calculated molecular mass of 12,473 Da and an electrophoretic mobility indistinguishable from that of the natural p15 subunit. When combined with the 110- and 18-kDa SIII subunits, bacterially expressed p15 efficiently replaces the natural p15 subunit in reconstitution of transcriptionally active SIII. A homology search revealed that the amino-terminal half of the SIII p15 subunit shares significant sequence similarity with a portion of the RNA-binding domain of Escherichia coli transcription termination protein rho and with the E. coli NusB protein, suggesting that SIII may be evolutionarily related to proteins involved in the control of transcription elongation in eubacteria.

RNA polymerase II transcription factor SIII. I. Identification, purification, and properties.

A transcription factor that stimulates synthesis of accurately initiated transcripts by RNA polymerase II has been identified and purified to apparent homogeneity from rat liver nuclear extracts. This factor, which we designate SIII, has a native molecular mass of approximately 140 kDa and is composed of three polypeptides of 110, 18, and 15 kDa. All three polypeptides are required to reconstitute SIII transcriptional activity. SIII appears distinct from previously identified mammalian transcription factors.

RNA polymerase II transcription factor SIII. II. Functional properties and role in RNA chain elongation.

A novel transcription factor that stimulates synthesis of accurately initiated transcripts by mammalian RNA polymerase II has been identified and purified to apparent homogeneity from rat liver extracts (Bradsher, J. N., Jackson, K. W., Conaway, R. C., Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). This factor, which we designate SIII, has a native molecular mass of approximately 140 kDa and is composed of three polypeptides of 110, 18, and 15 kDa. In this report, we demonstrate that SIII stimulates promoter-specific transcription by increasing the overall rate of RNA chain elongation by RNA polymerase II. Results of pulse-chase experiments indicate that SIII does not need to be present during preinitiation complex formation or transcription initiation in order to stimulate promoter-specific transcription. In addition, SIII is able to stimulate the rate of RNA chain elongation by RNA polymerase II during transcription of double stranded oligo(dC)-tailed templates. Taken together, these findings indicate that the factor exerts its activity directly on the elongation complex.

Transcription factor SIII: a novel component of the RNA polymerase II elongation complex.

A growing body of evidence suggests that the elongation stage of eukaryotic messenger RNA synthesis is a major site for the regulation of gene expression. In the process of investigating the mechanism of promoter-specific transcription by RNA polymerase II, we recently identified and purified a novel transcription factor that controls RNA chain elongation. This factor, which we have designated SIII, has a native molecular mass of 140 kDa and is composed of three polypeptides of 110, 18, and 15 kDa. Results of renaturation experiments indicate that all three polypeptides are required to reconstitute SIII transcriptional activity. A variety of evidence from mechanistic studies reveals that SIII dramatically stimulates the rate of RNA chain elongation by RNA polymerase II, most likely through a direct interaction with transcribing polymerase. Here we describe the properties of SIII as well as its role in regulating the activity of the RNA polymerase II elongation complex.

The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for function.

Transcription factor beta gamma (RAP30/74) from rat liver was previously shown in biochemical studies to control the binding of RNA polymerase II to promoters by a mechanism analogous to that utilized by bacterial sigma factors, by decreasing the affinity of polymerase for nonpromoter sites on DNA and by increasing the affinity of the enzyme for the preinitiation complex (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6205-6209). By constructing and analyzing mutants of beta gamma, we have identified a novel functional domain located in the carboxyl terminus of the gamma (RAP30) subunit. This domain shares sequence similarity with region 4 of bacterial sigma factors; in particular, it exhibits striking similarity to the carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis sigma k). Evidence from biochemical studies argues that a mutant gamma (RAP30), lacking amino acid sequences similar to sigma homology region 4.2, is able to assemble with the beta (RAP74) subunit to form a mutant beta gamma (RAP30/74) with impaired ability to interact with RNA polymerase II.

Mechanism of assembly of the RNA polymerase II preinitiation complex. Transcription factors delta and epsilon promote stable binding of the transcription apparatus to the initiator element.

Assembly of RNA polymerase II with the core region of TATA box-containing promoters requires the action of the TATA factor and four transcription factors designated alpha, beta gamma, delta, and epsilon, which have each been purified to near homogeneity from rat liver. Evidence from previous studies argues that alpha and beta gamma play a crucial role in delivering RNA polymerase II to the promoter (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6205-6209). Here we describe the interaction of transcription factor delta with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor tau from rat liver (Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. (1991) J. Biol. Chem. 266, 7804-7811). Results of template challenge experiments argue that delta enters the preinitiation complex through interactions with multiple components of the transcription apparatus. We observe that, in the presence of recombinant TFIID, delta interacts stably with the preinitiation complex only in the presence of RNA polymerase II, alpha, and beta gamma, whereas, in the presence of tau, delta is capable of interacting stably with the Initial Complex independently of RNA polymerase II. Results of restriction site protection experiments reveal that delta and epsilon promote binding of the transcription apparatus to the Initiator element and support the model that RNA polymerase II assembles at the core promoter in at least two discrete steps, first "touching down" near the TATA element and finally extending its interaction downstream to encompass the cap site.

Mechanism of assembly of the RNA polymerase II preinitiation complex. Evidence for a functional interaction between the carboxyl-terminal domain of the largest subunit of RNA polymerase II and a high molecular mass form of the TATA factor.

Genetic evidence argues that the highly conserved carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions directly in the regulation of transcription of many eukaryotic genes. The observation that partial deletion of the CTD of yeast RNA polymerase II reduces the ability of the enzyme to respond to signals from a variety of upstream activating sequences led to the proposal that the CTD plays a role in the dialogue between regulatory factors that bind upstream activating sequences and the "general" or "basal" transcription factors associated with RNA polymerase II at the promoter (Scafe, C., Chao, D., Lopes, J., Hirsch, J. P., Henry, S., and Young, R. A. (1990) Nature 347, 491-494). Biochemical evidence for an interaction of the CTD with specific components of the basal transcription apparatus, however, has been lacking. To identify target(s) for CTD action, we probed steps in assembly of the RNA polymerase II preinitiation complex with monoclonal antibodies specific for the CTD. Our findings reveal a novel interaction of the CTD with a high molecular mass form of the TATA factor. This interaction occurs during binding of RNA polymerase II to its promoter and requires the action of additional basal transcription factors; it is not observed when the single-subunit yeast transcription factor IID serves as the TATA factor.