We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy.

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position ؊3 to ؉6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position ؊6 to ؉6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position ؊19 to ؉8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.To counteract the detrimental effect of genotoxic attacks, cells activate sophisticated and specific DNA repair pathways. Damage induced by UV radiation, environmental agents, and anticancer drugs are removed by two distinct nucleotide excision repair (NER) 1 subpathways, namely global genome repair (GGR), which eliminates lesions from the entire genome, and transcription-coupled repair (TCR), a specialized pathway that repairs damages on a transcribed strand of active genes (1-3). Human NER involves the ordered action of factors in dual incision and DNA repair resynthesis steps (4). Any mutation that affects either the enzymatic activity or the ordered assembly of the dual incision complex leads to genetic disorders such as xeroderma pigmentosum, trichothiodystrophy, or Cockayne syndrome (5, 6).In global genome repair, the dual incision is a multistep process that results from the coordinated action of XPC/ HR23B, TFIIH, XPA, RPA, XPG, and XPF/ERCC1, resulting in the removal of the damaged oligonucleotide (4, 7, 8). After being recognized by the XPC/HR23B complex, the damaged DNA structure is targeted by TFIIH, which recruits the other factors upon the addition of ATP (9 -11). The unwound DNA is then incised by the two endonucleases XPG and XPF/ERCC1 on the 3Ј and 5Ј side of the lesion, respectively (12-15), leaving a gap structure that is filled up by the DNA polymerase ⑀ or ␦ and the accompanying factors PCNA, RF-C, RPA, and DNA ligase I (16). Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19). Although the hypothesis of the sequential assembly, which has gained a lot of support from recent biological studies, seems to be more accepted, the order of assembly of the NER factors on the damaged DNA and their contribution to the DNA remodeling to allow the repair are not fully understood (10,20,21). As an example, to further learn abou...

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.

The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein-DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions -40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.RNA polymerase II (RNAPII) is the multisubunit enzyme that synthesizes all mRNA precursors in eukaryotes. RNAPII is highly conserved among species, and in humans, RNAPII consists of 12 subunits, named Rpb1 to Rpb12 (16, 88). The two largest subunits, Rpb1 (220 kDa) and Rpb2 (140 kDa), form the enzyme's catalytic center and are homologous to the ␤Ј and ␤ subunits of bacterial RNAP, respectively. Five subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are also found in RNAPI and RNAPIII. Rpb3 and Rpb11 are homologous to the ␣ 2 homodimer involved in bacterial RNAP assembly. Rpb9 was attributed a role in elongation through its action at DNA arrest sites (3). In Saccharomyces cerevisiae, Rpb4 and Rpb7 form a subcomplex that can dissociate from the enzyme upon changes in environmental conditions. Under active growth conditions, most yeast RNAPII molecules do not contain the Rpb4-Rpb7 dimer, which primarily associates during the stationary phase or following stress (11). Functional studies of human RNAPII have been limited due to the lack of appropriate systems for purifying variant forms of the human enzyme.The availability of crystal structures of both yeast (17,18,26,30) and bacterial (8,57,58,84,89) RNAPs has been invaluable for understanding many of the molecular features of the transcription reaction. For example, the structure of elongating RNAPII has revealed the positioning of the RNA-DNA duplex during the transcription reaction (30). The structures available support a model in which the DNA enters the enzyme through a channel formed by a pair of "jaws" before accessing a deep cleft, at the bottom of which is buried the Mg 2ϩ -ion-containing active site; the DNA then turns by about 90°along a wall where the upstream end exits the enzyme (30). Many loops and helices either directly contact or closely approach the RNA-DNA duplex, thus suggesting putative functions for these structural elements in the transcription...

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.Initiation of mRNA synthesis by mammalian RNA polymerase II (pol II) is a complex biochemical process controlled by a set of general transcription factors including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1-5). Transcription initiation is preceded by the assembly of a preinitiation complex consisting of pol II and the general transcription factors on promoter DNA. For genes containing a TATA box, the first step in preinitiation complex assembly is the binding of TATA box-binding protein (TBP), the TATA box-binding subunit of TFIID, to the TATA element (6). The other general transcription factors and pol II can assemble, mostly through protein-protein interactions, onto the TBP-promoter complex (1-6).Human TFIIF, which is composed of two subunits known as RAP30 and RAP74, is involved at both the initiation and elongation stages of transcription (7). It is required for accurate initiation of transcription at all promoters tested except the IgH promoter (8-10). RAP30 is directly involved in recruiting pol II to a preinitiation complex containing TBP and TFIIB (11,12). TFIIF binds to several general initiation factors, including TFIIB (13-15), TFIID (16), and TFIIE (17), as well as to pol II (10,14,15,18). It is also an integral component of pol II holoenzymes isolated from both yeast and mammalian cells (19)(20)(21). These findings suggest that TFIIF plays a central role in preinitiation complex assembly. TFIIF is a target for at least some transcriptional activators, since the interaction of RAP74 with the serum response factor that binds the c-fos promoter is required for transcriptional activation (22,23). In addition, TFIIF can stimulate RNA chain elongation (7,24,25).Over the past few years, both high and low resolution techniques have been utilized to analyze the structure of the pol II preinitiation complex (26). NMR and x-ray crystallography studies revealed surprising features of TBP-promoter (27, 28), TBP-TFIIB-promoter (29, 30), and TBP-TFIIA-promoter (31, 32) complexes. Binding of TBP to the TATA element induces bending of the promoter DNA (27, 28). TFIIA and TFIIB both interact with TBP in the preinitiation complex, and TFIIB primarily by clamping onto the C-terminal stirrup of TBP (29, 30). Alanine scanning mutagenesis has identified regions ...