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The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription. Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription. At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH. However, in coding regions, the CTD is predominantly phosphorylated at Ser 2. Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb. Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation. Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase. Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation. Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5' end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.

The basal transcription factor TFIID consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Yeast Taf67 is homologous to mammalian TAF(II)55. Using a yeast two-hybrid screen to identify proteins that interact with Taf67, we isolated Bromodomain factor 1 (Bdf1) and its homolog (Bdf2). The Bdf proteins are genetically redundant, as cells are inviable without at least one of the two BDF genes. Both proteins contain two bromodomains, a motif found in several proteins involved in transcription and chromatin modification. The BDF genes interact genetically with TAF67. Furthermore, Bdf1 associates with TFIID and is recruited to a TATA-containing promoter. Deletion of Bdf1 or the Taf67 Bdf-interacting domain leads to defects in gene expression. Interestingly, the higher eukaryotic TAF(II)250 has an acetyltransferase activity, two bromodomains, and an associated kinase activity. Its yeast homolog, Taf145, has acetyltransferase activity but lacks the bromodomains and kinase. Bdf1, like TAF(II)250, has a kinase activity that maps carboxy-terminal to the bromodomains. The structural and functional similarities suggest that Bdf1 corresponds to the carboxy-terminal region of higher eukaryotic TAF(II)250 and that the interaction between TFIID and Bdf1 is important for proper gene expression.

A human cDNA was isolated encoding a protein with significant sequence similarity (41% identity) to the BVP RNA 5'-phosphatase from the Autographa californica nuclear polyhedrosis virus. This protein is a member of the protein-tyrosine phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998) J. Biol. Chem. 272, 20347-20353) to be a nuclear protein that can associate with RNA or ribonucleoprotein complexes. We demonstrate that PIR1 removes two phosphates from the 5'-triphosphate end of RNA, but not from mononucleotide triphosphates. The specific activity of PIR1 with RNA is several orders of magnitude greater than that with the best protein substrates examined, suggesting that RNA is its physiological substrate. A 120-amino acid segment C-terminal to the PTP domain is not required for RNA phosphatase activity. We propose that PIR1 and its closest homologs, which include the metazoan mRNA capping enzymes, constitute a subgroup of the PTP family that use RNA as a substrate.

Mutation of glutamate 62 to lysine in yeast transcription factor (TF) IIB (Sua7) causes a cold-sensitive phenotype. This mutant also leads to preferential transcription of downstream start sites on some promoters in vivo. To explore the molecular nature of these phenotypes, the TFIIB E62K mutant was characterized in vitro. The mutant interacts with TATA-binding protein normally. In three different assays, the mutant can also interact with RNA polymerase II and recruit it and the other basal transcription factors to a promoter. Despite the ability to assemble a transcription complex, the TFIIB E62K protein is severely defective in transcription in vitro. Therefore, the role of TFIIB must be more than simply bridging TATA-binding protein and polymerase at the promoter. We propose that the region around Glu-62 in yeast TFIIB plays a role in start site selection, perhaps mediating a conformational change in the polymerase or the DNA during the search for initiation sites. This step may be related to the yeast-specific spacing between TATA elements and start sites since mutations of the corresponding glutamate in mammalian TFIIB do not produce a similar effect.

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The yeast homolog of the mammalian RNA polymerase II general transcription factor TFIIA has been identified by complementation of a mammalian in vitro transcription system depleted for TFIIA. Like the mammalian factor, the yeast protein does not bind DNA, alters the size of the TFIID DNase I footprint at the adenovirus major late promoter, and forms specific TFIIA‐TFIID‐DNA complexes which are stable during electrophoresis in native acrylamide gels. The partially purified yeast factor was used to investigate its effect on the binding of TFIID to the major late promoter. Contrary to earlier models, we find that TFIIA does not significantly change the affinity or kinetics of TFIID binding, suggesting that it acts by altering the conformation of TFIID and/or by serving as a bridge between TFIID and the other general transcription factors.

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.

Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD-kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with high affinity to several TATA elements that do not match the consensus TATA sequences (TATAAA and TATATA): the yeast LEU2 TATA (TATTATTTA), the simian virus 40 TATA (CTTATTTAT), and the yeast CYC1 -10 TATA (TTATACATT) all bound TFIID. Furthermore, TFIID was active in promoting transcription in vitro from the nonconsensus TATA elements. Thus, contrary to previous suggestions, the existence of nonconsensus TATA elements does not itself indicate the existence of multiple TATA-binding factors.