Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase

In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ∼2.5-MDa (1) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2, 3). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4, 5). The 19 S regulatory particle consists of at least 17 different subunits (1, 6). Nine of these subunits form a “lid,” whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7–10) as well as cross-linking experiments (11, 12) have demonstrated that the six homologous ATPases are associated with the α rings of the 20 S particle.Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15). In both PAN and the 19 S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the α ring through which substrates enter the 20 S particle (15–17). Because this portal is quite narrow (18–20), only extended polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded by the associated ATPase complex to be translocated and digested within the 20 S particle.PAN and the six ATPases found at the base of the 19 S particle are members of the AAA+ superfamily of multimeric ATPases which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (8, 21). For mechanistic studies of the roles of ATP, the simpler archaeal PAN-20 S system offers many technical advantages over the much more complex 26 S proteasome. For example, prior studies of PAN (17, 22) demonstrated that unfolding of globular substrates (e.g. green fluorescent protein-ssrA) requires ATP hydrolysis. The same was also shown for the Escherichia coli ATP-dependent proteases ClpXP (23) and ClpAP (24). We have also shown that unfolding by PAN can take place on the surface of the ATPase ring in the absence of translocation (15). Thus, unfolding seems to proceed independently from protein translocation into the 20 S proteolytic particle. It is noteworthy that other studies suggest that proteins are unfolded by energy-dependent translocation through the ATPase ring (25, 26). These studies have suggested that the translocation of an unfolded polypeptide from the ATPase into the 20 S core is an active process that is coupled to ATP hydrolysis. A key to underline a detailed molecular mechanism for substrate binding, unfolding, and translocation by the proteasome regulatory ATPase complex is improved understanding of its architecture and the nucleotide-dependent structural transitions that afford these functions.To date we and others have failed to generate micrographs suitable for three-dimensional reconstruction of PAN using single-particle EM analysis. Likewise, structural information regarding the three-dimensional architecture and subunit organization within the 19 S particle is very limited. In fact, high resolution three-dimensional information on the 19 S complex is not yet available. Most knowledge available is based on cross-linking experiments (11, 12) as well as EM structural analysis (7–10), which provided a three-dimensional model outline of the general architecture of the 26 S complex. Unlike the 19 S complex, the structure of the 20 S subcomplex was determined by x-ray crystallography (18, 19). In contrast to the highly homogenous structure of the 20 S complex, the structural heterogeneity and flexibility of the 19 S subcomplex is presumably reflected in multiple conformations, which in turn also contribute to the difficulty in generating a high resolution three-dimensional structural model of the 26 S proteasome. Accordingly, the initial goal of this study was to generate a three-dimensional model of PAN that will allow us to determine its general architecture and to correlate unique conformational transitions within this ATPase with the nucleotide state of the complex (i.e. in the presence of ATPγS, ADP, or in the absence of nucleotides).Smith et al. (27) suggested a general architecture for the PAN-20 S complex based on two-dimensional averaging of a Thermoplasma acidophilum (TA)³ 20 S proteasome and Methanococcus jannaschii (MJ) PAN hybrid complex in the presence of ATPγS. Based on side-view projections of that complex, these authors proposed that PAN assumes an overall structure similar to E. coli HslU (28–30).We realized that although PAN appears heterogeneous in electron micrographs, it does not occupy all possible orientations when adsorbed to carbon-coated electron microscopy (EM) grids, a prerequisite for single particle analysis. This problem was overcome by applying electron tomography in conjunction with a three-dimensional averaging procedure that accounts for the missing wedge in the Fourier space of electron tomograms (31, 32). The three-dimensional model generated revealed an unexpected architecture leading to a possible molecular mechanism describing the function of PAN and presumably the 19 S ATPases.     

Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry

Ribosomal protein S1 has been shown to be a significant effector of prokaryotic translation. The protein is in fact capable of efficiently initiating translation, regardless of the presence of a Shine-Dalgarno sequence in mRNA. Structural insights into this process have remained elusive, as S1 is recalcitrant to traditional techniques of structural analysis, such as x-ray crystallography. Through the application of protein cross-linking and high resolution mass spectrometry, we have detailed the ribosomal binding site of S1 and have observed evidence of its dynamics. Our results support a previous hypothesis that S1 acts as the mRNA catching arm of the prokaryotic ribosome. We also demonstrate that in solution the major domains of the 30S subunit are remarkably flexible, capable of moving 30–50Å with respect to one another.Initiation of translation is often the rate-limiting step of protein biosynthesis (1). In prokaryotes, this process is widely recognized to be directed by the Shine-Dalgarno (S.D.)¹ sequence of mRNA and its complementation with the 3′ end of 16S rRNA (2). However, binding of the S.D. sequence to the ribosome is not obligatory for initiation. Ribosomal protein S1, widely conserved in prokaryotes, (3) has been shown to efficiently initiate translation, regardless of the presence of an S.D. sequence (4, 5).S1 is a strikingly atyptical ribosomal protein, being both the largest (61 kDa) and the most acidic (pI 4.7) (6). The protein is composed of six homologous repeats each forming beta barrel domains (3) that in solution comprise a highly elongated structure spanning up to ca. 230 Å (7). This length is comparable to the diameter of the ribosome itself. In addition to these anomalous characteristics, S1 is also one of only two ribosomal proteins that has been attributed functional significance (6). Ribosomal protein S1, for instance, has no apparent role in the assembly of the ribosome, (2) yet is critical for translation in E. coli (8, 9). The functional significance of S1 is related to its most pronounced characteristic, the ability to simultaneously bind mRNA and the ribosome. Analysis of fragments produced by limited proteolysis and chemical cleavage of S1 has shown that an N-terminal fragment of S1 (residues 1–193) binds the ribosome (10) but not RNA (11). Likewise, a C-terminal fragment (res 172–557) binds RNA (12, 13) but not the ribosome (6, 10). By nature of this bi-functional structure, S1 enhances the E. coli ribosome''s affinity for RNA ∼5000 fold (14) and can directly mediate initiation of translation by binding the 5′ UTR of mRNA (4, 5). These observations have led to the hypothesis that S1 acts as a catching arm for the prokaryotic ribosome, working to bring mRNA to the proximity of the ribosome and thereby facilitate initiation (6).Unfortunately, structural analyses capturing how S1 is able to function in this manner remain elusive. A high-resolution crystal structure of ribosome bound S1, or even free S1, does not exist, because S1 is recalcitrant to crystallography (6). Preparation of ribosomes for x-ray crystallography actually involves the deliberate removal of ribosomal protein S1 as a means to improve the reproducibility of crystallization and the quality of the ribosome crystals formed (15–17). The structure and interactions of the protein have nevertheless intrigued structural biologists for decades. However, studies completed to date have failed to convincingly demonstrate the interaction between S1 and the rest of the 30S subunit, because they were incapable of localizing the individual S1 domains (16, 18–20).We have studied the binding of S1 to the 30S subunit by combining cross-linking with mass spectrometry. Chemical cross-linking has long been appreciated as a technique to probe protein-protein interactions (21, 22). With the advent of modern mass spectrometers, it can be very effectively employed to confidently identify the exact residues involved in linkages (23–28). In most cross-linking analyses, protein residues are targeted for covalent modification with a molecule that contains two reactive groups separated by a spacer arm of known length. Only protein residues closer than the length of the spacer arm are capable of being linked. Identification of cross-linked residues thereby provides distance constraints for structural modeling. In this work, the novel amidinating protein cross-linker, DEST (diethyl suberthioimidate), was employed (29, 30). This amine reactive reagent, unlike commercially available reagents, preserves the native basicity of the residues it modifies while being effective at physiological pH. Use of the reagent is unlikely to perturb protein structure and the modifications it imparts are compatible with ionization for mass spectrometry. We have additionally shown that the cross-links it forms can be efficiently enriched from other components of proteolytic digests using strong cation exchange (SCX) chromatography, (30) and that DEST cross-linking of ribosomes yields structural information in excellent agreement with x-ray crystallography (29). Although DEST is an 11Å spacer arm cross-linker, it links alpha carbons up to 24Å apart because of the length and flexibility of lysine side chains. Nevertheless, this is sufficient resolution to approximate the binding positions of the 10kDa domains of S1. Furthermore, multiple cross-linking of a single domain significantly enhances the resolution with which it can be localized.Here, through the application of protein cross-linking and high resolution mass spectrometry, we show that S1 binds to the 30S subunit near the anti-S.D. motif of the 16S rRNA, demonstrate that it is highly elongated even when bound to the ribosome, and provide evidence that its C-terminal mRNA binding region is remarkably dynamic. Our results thus indicate S1 is structurally poised, as previously hypothesized, (6) to act as the mRNA catching arm of the prokaryotic ribosome.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS³ analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS³) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1–3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4–9). In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low resolution structural information about the constraints within a molecule (2, 3, 10) or protein complex (11–13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing the structural topology of proteins and/or protein complexes. Although this approach has been successful, effective detection and accurate identification of cross-linked peptides as well as unambiguous assignment of cross-linked sites remain extremely challenging due to their low abundance and complicated fragmentation behavior in MS analysis (2, 3, 10, 14). Therefore, new reagents and methods are urgently needed to allow unambiguous identification of cross-linked products and to improve the speed and accuracy of data analysis to facilitate its application in structural elucidation of large protein complexes.A number of approaches have been developed to facilitate MS detection of low abundance cross-linked peptides from complex mixtures. These include selective enrichment using affinity purification with biotinylated cross-linkers (15–17) and click chemistry with alkyne-tagged (18) or azide-tagged (19, 20) cross-linkers. In addition, Staudinger ligation has recently been shown to be effective for selective enrichment of azide-tagged cross-linked peptides (21). Apart from enrichment, detection of cross-linked peptides can be achieved by isotope-labeled (22–24), fluorescently labeled (25), and mass tag-labeled cross-linking reagents (16, 26). These methods can identify cross-linked peptides with MS analysis, but interpretation of the data generated from interlinked peptides (two peptides connected with the cross-link) by automated database searching remains difficult. Several bioinformatics tools have thus been developed to interpret MS/MS data and determine interlinked peptide sequences from complex mixtures (12, 14, 27–32). Although promising, further developments are still needed to make such data analyses as robust and reliable as analyzing MS/MS data of single peptide sequences using existing database searching tools (e.g. Protein Prospector, Mascot, or SEQUEST).Various types of cleavable cross-linkers with distinct chemical properties have been developed to facilitate MS identification and characterization of cross-linked peptides. These include UV photocleavable (33), chemical cleavable (19), isotopically coded cleavable (24), and MS-cleavable reagents (16, 26, 34–38). MS-cleavable cross-linkers have received considerable attention because the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis. Gas-phase cleavage sites result in the detection of a “reporter” ion (26), single peptide chain fragment ions (35–38), or both reporter and fragment ions (16, 34). In each case, further structural characterization of the peptide product ions generated during the cleavage reaction can be accomplished by subsequent MSⁿ1 analysis. Among these linkers, the “fixed charge” sulfonium ion-containing cross-linker developed by Lu et al. (37) appears to be the most attractive as it allows specific and selective fragmentation of cross-linked peptides regardless of their charge and amino acid composition based on their studies with model peptides.Despite the availability of multiple types of cleavable cross-linkers, most of the applications have been limited to the study of model peptides and single proteins. Additionally, complicated synthesis and fragmentation patterns have impeded most of the known MS-cleavable cross-linkers from wide adaptation by the community. Here we describe the design and characterization of a novel and simple MS-cleavable cross-linker, DSSO, and its application to model peptides and proteins and the yeast 20 S proteasome complex. In combination with new software developed for data integration, we were able to identify DSSO-cross-linked peptides from complex peptide mixtures with speed and accuracy. Given its effectiveness and simplicity, we anticipate a broader application of this MS-cleavable cross-linker in the study of structural topology of other protein complexes using cross-linking and mass spectrometry.     

Toward an Integrated Structural Model of the 26S Proteasome

The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.The ubiquitin-proteasome pathway is the major route used by eukaryotic cells for the disposal of misfolded or damaged proteins and for controlling the lifespan of proteins (1–3). As a consequence, the ubiquitin-proteasome pathway regulates a plethora of fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (4). The 26S proteasome is a molecular machine of ∼2.5 MDa that targets polyubiquitylated proteins. It comprises two subcomplexes, the 20S core particle (CP)¹ and one or two copies of asymmetric 19S regulatory particles (RPs), which bind to the end(s) of the barrel-shaped CP.The active sites of the proteasome are located in the CP cavity where proteolytic cleavage of substrates takes place. Electron microscopy (EM) and x-ray crystallography have revealed that the CP is a cylinder consisting of four concentrically stacked rings (5–7): two identical “α”-rings, each assembled of seven homologous proteins, form the outer rings, and two identical “β”-rings, also assembled of seven homologs, form the two inner rings. Proteolysis is confined to the cavity formed by the β-rings, a nanocompartment sequestered from the cytosol.The RPs regulate substrate degradation by (i) binding polyubiquitylated substrates, (ii) subsequently deubiquitylating them, (iii) substrate unfolding, and (iv) opening the “gate” to the CP (8). The RPs consists of six AAA-ATPase subunits and at least 13 non-ATPase subunits. In contrast to the CP, the architecture of the RP subunits remains largely unknown. The problems that hamper structural characterization of the RP are manifold. It has proven difficult to obtain homogeneous, concentrated preparations of 26S proteasomes or RPs because the RP tends to disassociate into heterogeneous subcomplexes during purification and concentration. Moreover, many of the RP subunits likely exhibit a significant degree of structural variability. As a consequence, x-ray crystallographic analysis of the entire RP has not been accomplished to date, and only a few subunit fragments have been amenable to high resolution structure determination.For cryo-EM and protein-protein interaction experiments, the requirements for sample homogeneity are less stringent. Recently, the Drosophila melanogaster 26S proteasome was resolved to ∼20 Å (9). Various proteomics approaches have led to proposals for topological maps of the RP (10–13). The resolution of protein-protein interaction data typically corresponds to the diameters of the proteins or domains found to interact, which are typically far beyond 20 Å. Because of the limited resolution, neither cryo-EM maps nor protein-protein interaction networks are by themselves sufficient to determine the RP architecture (i.e. the localization of the RP subunits in the complex).The integration of atomic models, cryo-EM maps, and protein-protein interaction data is currently the most promising approach to resolve the architecture of the 26S proteasome (14–18). Here, we provide a comprehensive analysis of the current structural knowledge on the RP, including structures of RP subunits, physical protein-protein interactions, and cryo-EM data. Based on these data, we provide a model for the CP-AAA-ATPase subcomplex. Finally, we outline a path toward resolving the architecture of the 26S proteasome by an integrative structure determination approach, which in turn will provide a basis for a mechanistic understanding.     

Subcomplexes of PA700, the 19 S Regulator of the 26 S Proteasome, Reveal Relative Roles of AAA Subunits in 26 S Proteasome Assembly and Activation and ATPase Activity

We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1–6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.The 26 S proteasome is a 2,500,000-Da protease complex that degrades polyubiquitylated proteins by an ATP-dependent mechanism (1, 2). The biochemical processes required for this function are divided between two subcomplexes that compose the holoenzyme (3, 4). The first, called 20 S proteasome or core particle, is a 700,000-Da complex that catalyzes peptide bond hydrolysis (5). The second, called PA700 or 19 S regulatory particle, is a 700,000-Da complex that mediates multiple aspects of proteasome function related to initial binding and subsequent delivery of substrates to the catalytic sites of the 20 S proteasome (6). The 20 S proteasome is composed of 28 subunits representing the products of 14 genes arranged in four axially stacked heteroheptameric rings (7, 8). Each of the two center β rings contains three different protease subunits that utilize N-terminal threonine residues as catalytic nucleophiles (5, 8, 9). These residues line an interior lumen formed by the stacked rings and thus are sequestered from interaction with substrates by a shell of 20 S proteasome subunits.PA700 is composed of 20 different subunits. Six of these subunits, termed Rpt1–6, are AAA² (ATPases Associated with various cellular Activities) family members that confer ATPase activity to the complex and mediate energy-dependent proteolysis by the 26 S proteasome (2, 10). 26 S proteasome assembly from PA700 and 20 S proteasome requires ATP binding to Rpt subunits (11–15). Binding of PA700 to the 20 S proteasome occurs at an axial interface between a heterohexameric ring of the PA700 Rpt subunits and the heteroheptameric outer ring of α-type 20 S proteasome subunits (16). Substrates enter the proteasome through a pore in the center of the α subunit ring that is reversibly gated by conformationally variable N-terminal residues of certain α subunits in response to PA700 binding (12, 17–19). Although the degradation of polyubiquitylated proteins requires additional ATP hydrolysis-dependent actions by PA700, the assembled 26 S proteasome displays greatly increased rates of energy-independent degradation of short peptides by virtue of their increased access to catalytic sites via diffusion through the open pore (15, 18, 20).Recently, specific interactions between Rpt and α subunits that determine PA700-20 S proteasome binding and gate opening have been defined. These findings established nonequivalent roles among the six different Rpt subunits for these processes (12, 19). For example, carboxypeptidase A treatment of PA700 selectively cleaves the C termini of two Rpt subunits (Rpt2 and Rpt5) and renders PA700 incompetent for proteasome binding and activation (19). Remarkably, short peptides corresponding to the C terminus of either Rpt2 or Rpt5, but none of the other Rpt subunits, were sufficient to bind to the 20 S proteasome and activate peptide substrate hydrolysis by inducing gate opening (12, 15, 18). The C-terminal peptides of Rpt2 and Rpt5 appear to bind to different and distinct sites on the proteasome and produce additive effects on rates of peptide substrate hydrolysis, suggesting that pore size or another feature of gating can be variably modulated (19). These various results, however, do not specify whether the action of one or the other or both C-terminal peptides is essential for function of intact PA700.In addition to its role in activation, PA700 plays other essential roles in 26 S proteasome function related to substrate selection and processing. For example, PA700 captures polyubiquitylated proteins via multiple subunits that bind polyubiquitin chains (21–23). Moreover, to ensure translocation of the bound ubiquitylated protein through the narrow opened substrate access pore for proteolysis, PA700 destabilizes the tertiary structure of the protein via chaperone-like activity and removes polyubiquitin chains via deubiquitylating activities of several different subunits (24–30). These various functions appear to be highly coordinated and may be mechanistically linked to one another and to the hydrolysis of ATP by Rpt subunits during substrate processing.Despite support for this general model of PA700 action, there is a lack of detailed knowledge about how PA700 subunits are structurally organized and functionally linked. Previously, we identified and characterized a subcomplex of PA700 called “modulator” that contained two ATPase subunits, Rpt4 and Rpt5, and one non-ATPase subunit, p27 (31). Although this protein was identified by an assay that measured increased PA700-dependent proteasome activation, the mechanistic basis of this effect was not clear. Moreover, the modulator lacked detectable ATPase activity and proteasome activating activity. The latter feature is surprising in retrospect because of the newly identified capacity of Rpt5 to activate the proteasome directly (12, 19). This disparity suggests that specific interactions among multiple PA700 subunits determine the manifestation and regulation of various activities.This study extends our recent findings regarding relative roles of Rpt subunits in the regulation of proteasome function. It also provides new insights and significance to older work that identified and characterized the modulator as a subcomplex of PA700. Our findings unite two different lines of investigation to offer new information about the structure, function, and regulation of 26 S proteasome. They also offer insights about alternative models for assembly of PA700 and 26 S proteasome in intact cells.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K_m value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.Sphingosine 1-phosphate (S1P),² a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P₁-S1P₅) have been identified in endothelial and immune cells (4). Studies using S1P₁ receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P₁ receptor on cell surfaces migrate (2, 5, 6, 13–15).The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca²⁺ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes.     

Heparan Sulfate Proteoglycans Are Receptors for the Cell-surface Trafficking and Biological Activity of Transglutaminase-2

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.Transglutaminase type 2 (TG2,² EC 2.3.2.13) is the most widespread member of a large family of enzymes that catalyze the Ca²⁺-dependent post-translational modification of proteins leading to intra- or intermolecular Nϵ(γ-glutamyl)lysine bonds (1, 2). Unlike other family members, TG2 is uniquely exported through a yet to be elucidated non-conventional pathway. Once secreted, TG2 finds in the extracellular compartment the ideal conditions of high Ca²⁺ and low GTP concentration for the activation of its intrinsic transamidation activity (cross-linking) (2, 3). Intracellularly, GTP binding suppresses the Ca²⁺-dependent cross-linking activity and determines the additional GTPase activity of TG2 (4, 5), which is responsible for signal transduction (6). Once externalized, TG2 remains tightly bound to the cell surface and to the extracellular matrix (ECM) (7, 8), and it is rarely found free in the conditioned medium, unless overexpressed by cell transfection (9).Extracellular TG2 activity is involved in the cross-linking of the ECM, conferring resistance to matrix metalloproteinase and promoting cell-matrix interactions via cross-linking of fibronectin (FN) and collagen (1, 7, 11, 12). TG2 has an additional non-enzymatic role in the matrix as an integrin-β₁ co-receptor (8) by supporting RGD-independent cell adhesion to FN (8, 13, 14).Extracellular cross-linking and TG2-mediated adhesion facilitate the repair process in many tissue compartments (1, 2, 15, 16). On the other hand, uncontrolled cross-linking as a consequence of chronic cell insult and secretion of TG2 has been implicated in a number of pathological conditions, including kidney, liver, and pulmonary fibrosis (17–20).Understanding how TG2 is exported and targeted to the cell surface is critical for limiting its cellular secretion and extracellular action. Although a key trigger for TG2 export is cell stress (2, 21, 22), TG2 is not unspecifically released, because extracellular trafficking occurs in the absence of leakage of intracellular components and cells remain viable (23). We know that TG2 requires the tertiary structure of its active site region to be secreted (9); moreover, TG2 is acetylated on the N terminus (24), a process reported to affect membrane targeting of non-conventional secreted proteins (25). Two main binding partners for TG2, FN and integrin-β1, have both been attributed a possible role in the transport of TG2 to the cell surface (8, 26). FN was shown to co-localize with TG2 once released (26), and integrin-β1 to co-associate with TG2 in cells induced to differentiate (8).TG2 has also long been known to have some affinity for heparin (27, 28), a highly sulfated analogue of heparan sulfate (HS) glycosaminoglycan chains, which are abundant constituents of the cell surface/ECM. HS chains are linear polysaccharides consisting of alternating N-acetylated or N-sulfated glucosamine units (GlcNAc or GlcNS), and uronic acids (glucuronic acid GlcA or iduronic acid IdoA residues) (29), which only exist covalently bound to the core protein of cell-surface proteoglycans (syndecans and glypicans) and secreted proteoglycans (29). Heparin binding is a property common to many ECM proteins (29), but the level of affinity has never been established for TG2, which makes it difficult to estimate the real biological significance of this interaction. Heparan sulfate proteoglycans (HSPG) bind ECM ligands through the HS chains, influencing their biological activity, trafficking, and secretion. Among the HSPG subfamilies, the syndecans act as co-receptors for both ECM components and soluble ligands (30), and syndecan-4 has overlapping roles with extracellular TG2 in wound healing and fibrosis (31, 32). In this study, we show that TG2 has a surprisingly high affinity for heparin and HS, raising the hypothesis that HSPG are involved in its biological activity. We demonstrate that HSPGs are essential for the transamidating activity of TG2 at the cell surface and that syndecan-4 acts as a receptor for TG2, which is involved in the trafficking and cell-surface localization, and thus activity of TG2.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Dithiothreitol (DTT) Acts as a Specific,UV-inducible Cross-linker in Elucidation of Protein–RNA Interactions*

Protein–RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein–RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein–RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C₄H₈S₂O₂) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.Cross-linking of biomolecules combined with mass spectrometry (MS) has emerged as a powerful tool to characterize not only the tertiary and quaternary arrangements of individual biomolecules, but especially their interaction sites in biologically active complexes. By MS-based identification of the cross-linked parts or even the exact cross-linking sites of the respective biomolecules, proximity information can be derived. This has proven highly useful for computational approaches to problems such as docking or the arrangement of subunits (1–3).In principle, cross-linking can be achieved in two ways: (1) By using a chemical cross-linker that connects reactive groups of the respective biomolecules within a certain distance range, the range depending on the reagent used. (2) By generating a so-called zero-length cross-link that connects reactive groups of biomolecules that are already directly adjacent to one another. The latter is usually achieved by (UV) light-induced cross-linking, with or without the addition of compounds that induce the generation of radicals on reactive groups of the cross-linkable components or in close vicinity to them.Cross-linking in combination with MS analysis is nowadays frequently used in protein-protein interaction studies (4–7) but can also be applied to protein-nucleic acid complexes. Indeed much attention is currently paid to their MS-based analysis owing to the crucial cellular function of many such complexes. A large variety of studies over decades have examined chemical cross-linking between proteins and DNA, using reagents such as the genotoxic diepoxybutane, endogenous aldehydes, transition metals, nitrogen mustard, and platinum compounds, alkynitrosoureas, and formaldehyde (8). In addition, UV irradiation has been used to establish cross-links between proteins and protein-bound single-stranded DNA (ssDNA), which can then be identified by mass spectrometry (9, 10). UV cross-linking makes use of the natural sensitivity of nucleobases to UV light (11, 12). The site of cross-linking can then be determined by mass spectrometric analysis (including gas phase fragmentation of the corresponding peptide-nucleic acid conjugates) and database searching. To enhance the low yield of protein-DNA cross-linking, derivatives with higher UV reactivity, such as halonucleotides, can be employed (13–15).UV-induced cross-linking is more frequently used to monitor protein–RNA interactions. RNA is mainly present in its single-stranded form, and adopts a variety of tertiary structures in which the bases of the nucleotides are in close contact with amino acid residues of the proteins that are bound to the RNA. Several studies have used this technique to identify globally the proteins that directly interact with e.g. poly(A) mRNA in yeast and human systems, and have yielded insight into the large variety of RNA-binding proteins that exist within the cell (16–21). More detailed approaches apply UV-induced protein–RNA cross-linking in a similar manner, but extend the MS analysis toward the identification of the cross-linked amino acids together with the corresponding nucleotide moieties, allowing exact definition of the RNA-binding regions in the cross-linked proteins (22–24). To improve the yield of cross-linking, more reactive nucleoside derivatives (4-thiouridine and 6-thioguanosine) have been incorporated into RNA in growing cells. The cross-linking reaction of these derivatives with amino acids is not entirely additive, however, but is accompanied by the loss of H₂S ((22), U.Z. and H.U, unpublished results). Other cross-linking reactions between proteins and RNA have made use of nucleotide derivatives that contain a cross-linkable function at the 2′ hydroxyl group or the phosphate moiety (25–27), but have not been characterized by MS yet.Additional cross-linking agents for the analysis of protein–RNA interactions have been exploited, but have not yet found their way into modern MS-based proteome analyses. For instance, methylene blue has been described as a light-inducible cross-linker, in particular for mapping interactions of proteins with dsRNA (28). Similarly, protein–RNA interaction studies in ribosomal subunits have made use of diepoxybutane, or nitrogen mustard (29–31). The same holds true for the use of 2-iminothiolane (“Traut''s reagent”), which is a protein–RNA cross-linking reagent that combines chemical and UV-inducible features and has been extensively applied to the analysis of protein–RNA contacts in ribosomal subunits (33, 32). Here, we introduce dithiothreitol (DTT)¹ as a potent UV-inducible cross-linking reagent for the analysis of protein–RNA linkages following UV irradiation. By exhaustive mass-spectrometric analyses we found that upon UV light exposure DTT forms a covalent linkage between cysteine residues within proteins and uracil bases of RNA in close proximity. We applied this to protein–RNA complexes isolated from yeast cells and compared the protein–RNA cross-linking patterns that were obtained in the presence and absence of DTT. We found that the cross-linking reaction is surprisingly efficient and specific.