Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.

Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5¢ end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.In recent years, high-resolution structural models of large macromolecular complexes such as the ribosome 1 , the RecBCD helicase 2 or RNA polymerases 3,4 have been obtained using X-ray crystallography. Although these structures provide detailed insight into the molecular architecture of complex biological systems, the position of flexible domains can usually not be determined because of averaging effects.Single-molecule methods, on the other hand, provide the possibility of directly obtaining structural information because they allow the study of real-time conformational changes of macromolecular complexes 5 . In combination with fluorescence resonance energy transfer (FRET) 6 , a technique that has been termed a molecular ruler 7 , one can in principle measure distances within a macromolecule in real-time. However, because of experimental problems such as variations in quantum yield 8 or dependence of FRET on the orientations of the two dye molecules 9 , there are few examples in the literature of quantitative distance measurements 8,10-12 or position determination 13-16 using single-pair FRET (sp-FRET). Instead, these data are more often interpreted in a qualitative fashion monitoring conformational changes and length increases or decreases [17][18][19][20] .Using triangulation of several FRET distance measurements, it is possible to determine a previously unknown position [13][14][15][16][21][22][23] . Although these experiments are able to estimate the most likely position, they do not show how existing experimental uncertainties might affect the position determined. Therefore, these positions must be interpreted with great caution because one has no information about the experimental accuracy. In principle, one can conduct control measurements that provide validity tests of the position determined 14 , but to arrive at a quantitative technique, experimental uncertainties must be taken into account.Here we used bayesian parameter estimation 21 , a probabilitybased analysis method, to compute the three-dimen...

The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.

The incidence and death rate of pancreatic ductal adenocarcinoma (PDAC) have increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumour formation and development of metastasis. Here we report that PDAC cells secrete BAG3, which binds and activates macrophages, inducing their activation and the secretion of PDAC supporting factors. We also identify IFITM-2 as a BAG3 receptor and show that it signals through PI3K and the p38 MAPK pathways. Finally, we show that the use of an anti-BAG3 antibody results in reduced tumour growth and prevents metastasis formation in three different mouse models. In conclusion, we identify a paracrine loop involved in PDAC growth and metastatic spreading, and show that an anti-BAG3 antibody has therapeutic potential.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

To further our understanding of the transcription/ DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstituted in vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the Cterminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD.Human TFIIH is a multiprotein complex composed of nine subunits ranging from 89 to 32 kDa. These subunits are assembled into two subcomplexes: the core TFIIH, which is composed of six subunits (XPB, XPD, p62, p52, p44, and p34) and the cdk-activating kinase (CAK), 1 which is composed of cdk7, cyclin H, and MAT1. Recently, the molecular structures of both human and yeast TFIIHs have been determined by electron microscopy and show similarities in size, shape, and architecture (1, 2). Originally identified as a basal transcription factor, TFIIH was subsequently found to contain XPB (Rad 25) and XPD (Rad 3), two helicases involved in nucleotide excision repair (NER) (3-8). Mutations in one of these subunits induce UV sensitivity in both human and yeast and are responsible for three rare human genetic disorders, xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy (9, 10). Many XP patients suffer from a high incidence of skin cancer due to their inability to remove lesions from their DNA. Further studies in yeast have shown that additional subunits of TFIIH, including p62 (Tfb1), p52 (Tfb2), and p44 (Ssl1) also have an essential role in DNA repair (11).In an effort to understand the function of TFIIH in the diverse fundamental cellular processes in which it participates (transcription and DNA repair but also cell cycle regulation through the CAK complex), efforts have been aimed at systematically characterizing all TFIIH subunits. Several functions have been determined for individual components: XPB and XPD are ATP-dependent helicases indispensable for opening the DNA around a promoter and/or a lesion (12-16). Cdk7 is a serine/threonine kinase, which is regulated by cyclin H and MATI and phosphorylates several substrates including the Cterminal domain (CTD) of RNA polymerase II (see Ref. 17 and references therein). Recently, the N-terminal part of p44, a subunit of core TFIIH, has been shown to positively regulate XPD helicase activity, whereas the C-terminal part is involved in promoter escape (18). The XPD helicase regulation is lacking in a majority of XP-D patients because mutations in the Cter...

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.