Transcription Initiation Process Research Articles

Alteration of the -35 and -10 sequences and deletion the upstream sequence of the -35 region of the promoter A1 of the phage T7 in dsDNA confirm the contribution of non-specific interactions with E. coli RNA polymerase to the transcription initiation process.

Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the -10 and -35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements -35 and -10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the -35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region -35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered -35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also that the process of promoter localization and recognition, as well as the formation of binary complexes, is sequential in nature, and that the region located upstream of the -35 hexamer, and the hexamer itself, plays here an additive role.

Mechanisms of Honeysuckle Against Triple-negative Breast Cancer Based on Network Pharmacology and Molecular Docking

[Objective] To study the mechanism of action of the Honeysuckle against triple-negative breast cancer based on network pharmacology and molecular docking method, to provide a reference for elucidating its clinical anti-breast cancer mechanism. [Methods] The Honeysuckle active ingredients and their targets were screened by TCMSP and SwissTargetPrediction databases, and the target genes related to breast cancer were collected by GeneCards, OMIM and Disgenet databases, and the "Drug-Active Ingredient-Target-Disease" was constructed by using Cytoscape 3.9.1 software. The Cytoscape 3.9.1 software was used to construct a "drug-active ingredient-target-disease" network. The potential targets were imported into the STRING database to build a protein-protein interactions network (PPI). The core targets were annotated with the GO function and analyzed by KEGG pathway enrichment through the DAVID database. Molecular docking was carried out between the main active ingredients of the Honeysuckle and the potential targets through the Pymol software and AutoDock software. Molecular docking was performed using Pymol and AutoDock software. [A total of 23 active ingredients and 591 targets of the Honeysuckle, 1,811 triple-negative breast cancer disease targets, 186 potential targets, and 38 genes in the PPI network were screened in the two-component target databases, TCMSP and SwissTargetPrediction, and the three disease databases, GeneCards, OMIM, and Disgenet. In the PPI network, 38 key targets of the honeysuckle for breast cancer treatment were obtained, and after GO and KEGG analysis, 1,118 GO analysis results and 157 KEGG pathways were obtained. The GO analysis results showed that the honeysuckle's anti-breast cancer may be related to the transcription initiation process of the RNA polymerase II promoter, nuclear chromatin, DNA binding, etc.; the KEGG analysis results showed that the honeysuckle may be involved in the cancer pathway and the anti-breast cancer may involve the cancer pathway. The results of the KEGG analysis indicated that the honeysuckle anti-breast cancer might be involved in the cancer pathway, proteoglycan signaling pathway, microRNA pathway, thyroid hormone signaling pathway, etc. The molecular docking results showed that the predicted key components, quercetin and lignocerol, had better binding properties to the core targets, EP000, ERBB2, GAPDH, and MAPK3. [Conclusion] The anti-triple-negative breast cancer of the Honeysuckle mainly involves EP000, ERBB2, GAPDH , MAPK3 and 157 signaling pathways, and its active ingredients can inhibit the growth of tumor cells through multi-targets, multi-pathways, and multi-mechanisms.

Fe(II) oxygenation inhibits bacterial Mn(II) oxidation by P. putida MnB1 in groundwater under O2-perturbed conditions

Bacterial Mn(II) oxidation plays a crucial role in Mn cycling and the associated biogeochemistry in natural waters and is of practical concern in the clean-up of excess Mn from drinking water. Fe(II) usually occurring together with Mn(II) in groundwater is oxidized prior to Mn(II) when perturbed by O2, but the impact of Fe(II) oxygenation on the subsequent bacterial Mn(II) oxidation remains unknown. Here we demonstrated that Fe(II) oxygenation largely inhibited the Mn(II)-oxidizing ability of MnB1 belong to Pseudomonas putida which is ubiquitous in groundwater. The mechanisms of the inhibition varied under different Fe(II) concentrations. At high Fe(II) concentrations (≥ 1 mM), the inhibition of bacterial Mn(II) oxidation was mainly because of cell death caused by intracelluar reactive oxygen species. At low Fe(II) concentrations (≤ 0.05 mM), the inhibition of bacterial Mn(II) oxidation was attributed to Fe(III) oxyhydroxides generated from Fe(II) oxygenation. Fe(III) oxyhydroxides attached to cell surface and damaged the cell membrane, resulting in the influx of dissolved Fe into the cell. Transcriptomic analysis revealed that the intracellular Fe suppressed the transcription initiation process and the subsequent generation of multicopper oxidases which were responsible for Mn(II) oxidation. These findings implicate that the inhibition effect of Fe(II) oxygenation on bacterial Mn(II) oxidation should be considered in groundwater-surface water interaction zone and the biological treatment of Fe-Mn containing drinking water.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

In silico prediction of secretory proteins of Opisthorchis viverrini, Clonorchis sinensis and Fasciola hepatica that target the host cell nucleus

Liver flukes Fasciola hepatica, Opisthorchis viverrini and Clonorchis sinensis are causing agents of liver and hepatobiliary diseases. A remarkable difference between such worms is the fact that O. viverrini and C. sinensis are carcinogenic organisms whereas F. hepatica is not carcinogenic. The release of secretory factors by carcinogenic flukes seems to contribute to cancer development however if some of these target the host cell nuclei is unknown. We investigated the existence of O. viverrini and C. sinensis secretory proteins that target the nucleus of host cells and compared these with the corresponding proteins predicted in F. hepatica. Here we applied an algorithm composed by in silico approaches that screened and analyzed the potential genes predicted from genomes of liver flukes. We found 31 and 22 secretory proteins that target the nucleus of host cells in O. viverrini and C. sinensis, respectively, and that have no homologs in F. hepatica. These polypeptides have enriched the transcription initiation process and nucleic acid binding in O. viverrini and C. sinensis, respectively. In addition, other 11 secretory proteins of O. viverrini and C. sinensis, that target the nucleus of host cells, had F. hepatica homologs, have enriched RNA processing function. In conclusion, O. viverrini and C. sinensis have 31 and 22 genes, respectively, that may be involved in their carcinogenic action through a direct targeting on the host cell nuclei.

Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe.

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Probing Mechanisms of Transcription Elongation Through Cell-to-Cell Variability of RNA Polymerase

The process of transcription initiation and elongation are primary points of control in the regulation of gene expression. Although biochemical studies have uncovered the mechanisms involved in controlling transcription at each step, how these mechanisms manifest in vivo at the level of individual genes is still unclear. Recent experimental advances have enabled single-cell measurements of RNA polymerase (RNAP) molecules engaged in the process of transcribing a gene of interest. In this article, we use Gillespie simulations to show that measurements of cell-to-cell variability of RNAP numbers and interpolymerase distances can reveal the prevailing mode of regulation of a given gene. Mechanisms of regulation at each step, from initiation to elongation dynamics, produce qualitatively distinct signatures, which can further be used to discern between them. Most intriguingly, depending on the initiation kinetics, stochastic elongation can either enhance or suppress cell-to-cell variability at the RNAP level. To demonstrate the value of this framework, we analyze RNAP number distribution data for ribosomal genes in Saccharomyces cerevisiae from three previously published studies and show that this approach provides crucial mechanistic insights into the transcriptional regulation of these genes.

Near-atomic resolution visualization of human transcription promoter opening

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA-RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

Structural Insights into the Eukaryotic Transcription Initiation Machinery.

Eukaryotic gene transcription requires the assembly at the promoter of a large preinitiation complex (PIC) that includes RNA polymerase II (Pol II) and the general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. The size and complexity of Pol II, TFIID, and TFIIH have precluded their reconstitution from heterologous systems, and purification relies on scarce endogenous sources. Together with their conformational flexibility and the transient nature of their interactions, these limitations had precluded structural characterization of the PIC. In the last few years, however, progress in cryo-electron microscopy (cryo-EM) has made possible the visualization, at increasingly better resolution, of large PIC assemblies in different functional states. These structures can now be interpreted in near-atomic detail and provide an exciting structural framework for past and future functional studies, giving us unique mechanistic insight into the complex process of transcription initiation.

Near-atomic resolution visualization of human transcription promoter opening.

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA-RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

Where does transcription start? 5'-RACE adapted to next-generation sequencing.

The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5′ cDNA ends (5′-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5′-m7G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R) and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5′ m7G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5′ UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GR N-terminal protein isoform levels.

Estimation of kinetic parameters of transcription from temporal single-RNA measurements

Gene expression dynamics in prokaryotes is largely controlled by the multi-step process of transcription initiation whose kinetics is subject to regulation. Since the number and duration of these steps cannot be currently measured in vivo, we propose a novel method for estimating them from time series of RNA numbers in individual cells.We demonstrate the method’s applicability on measurements of fluorescence-tagged RNA molecules in Escherichia coli cells, and compare with a previous method. We show that the results of the two methods agree for equal data. We also show that, when incorporating additional data, the new method produces significantly different estimates, which are in closer agreement with qPCR measurements. Unlike the previous method, the new method requires no preprocessing of the RNA numbers, using maximal information from the RNA time series. In addition, it can use data outside of the observed RNA productions. Overall, the new method characterizes the transcription initiation process with enhanced detail.

Functional reconstitution of an unusual Firmicutes σ factor into a Gram-negative heterologous host

Sigma (σ) factors are single-subunit proteins that reversibly bind RNA polymerase and play an important role in the transcription initiation process. An unusual 2-subunit σ factor, consisting of proteins SigO and RsoA, activates transcription from a group of related promoters in Bacillus subtilis. These 2 proteins specifically interact with each other and with RNA polymerase subunits. This system is widespread among species in several Bacillus-related genera, but otherwise appears restricted to the Firmicutes. Here, we reconstituted SigO-RsoA, and a cognate promoter, into the distantly related heterologous host Escherichia coli to examine whether this system can function in bacteria outside of the Firmicutes. We show that these proteins can productively associate with E. coli RNA polymerase and activate transcription, demonstrating that there are no structural barriers to function. In parallel, we tested a wide array of protein-protein interaction mutations and promoter mutations that impact SigO-RsoA function in both B. subtilis and E. coli and conclude that the SigO-RsoA system behaves, in most instances, similarly in both genetic backgrounds. These data raise the possibility of genetically isolating the system in this heterologous host and away from unknown B. subtilis factors that may also be playing a role in SigO-RsoA regulatory pathways, thus facilitating further study of the system. As a result of this work, we also provide a comprehensive mutational analysis of a SigO-RsoA promoter and report the preliminary identification of amino acids in SigO that play a role in mediating the SigO-RsoA protein-protein interaction.

Uncoupling Promoter Opening from Start-Site Scanning

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.

Structural studies of Mot1 and NC2 in transcription initiation process

Regulation of protein–nucleic acid interactions plays a key role in various cellular processes. The SNF2/SWI2 protein family forms a large and diverse class of proteins and multiprotein assemblies, which use energy derived from ATP hydrolysis to disrupt or modify protein-DNA interactions. They possess a conserved helicase-like domain accompanied by protein-specific targeting and regulation regions. The SNF2/SWI2 family member Mot1 (Modifier of transcription 1, also known as BTAF1) is a single polypeptide ATP-dependent remodeler. Mot1 acts directly on TBP (TATA-box binding protein) regulating RNA polymerase II preinitiation complex formation in the first stages. Global distribution of TBP on promoter regions is also modulated by NC2 (Negative Cofactor 2). It has been suggested that Mot1 and NC2 can co occupy the same promoter sites influencing the assembly of the transcription machinery simultaneously. Our understanding of the molecular mechanism of SWI2/SNF2 family ATPases is very limited. Previously, we have reported the crystal structure of the complex of Encephalitozoon cuniculi N terminal domain of Mot1 (Mot1NTD) with its substrate, TBP [1]. Here we present the crystal structure of the TBP NC2 Mot1NTD complex bound to a promoter DNA fragment at 3.9Å resolution. In our studies we have applied a combined structural biology approach using crystallography, electron microscopy reconstructions and chemical cross-linking. We probed the conformational changes of the complex during the ATP hydrolysis cycle and unveiled the structural basis of the Mot1–NC2 interplay. Our findings greatly contribute not only to our limited understanding of Mot1 action, but all SNF2/SWI2 family remodeling enzymes.

Structural insight into HIV-1 reverse transcription initiation in MAL-like templates (CRF01_AE, subtype G and CRF02_AG)

Based on the known structural model for reverse transcription initiation complex of the human immunodeficiency virus type 1 (HIV-1) MAL isolate, we attempted to predict a structural behavior of MAL-like templates (CRF01_AE, subtype G and CRF02_AG) within the initiation complex by in silico experiments. Switches from the D-duplex (dimerization-competent) conformation to the I-duplex (initiation-competent) conformation and then to conformations with an open primer activation signal (PAS) structure have been examined for four fragments of U5 and primer binding site (PBS) region, the minimal fragment (nt 121–243), fragment 1 (nt 110–243), fragment 2 (nt 113–259), and extended fragment 2 (nt 109–261). Switches from the D-duplex conformation to the I-duplex conformation in the minimal fragment or fragment 1 and from the I-duplex conformation to conformations with exposed PAS motif in fragment 1 are similar in all MAL-like templates. A PAS exposure in fragment 2 and extended fragment 2 is supported by PBS stem extension which structure is affected by subtype-specific variations in CRF01_AE (the mutated motif 116GUUAG120) and CRF02_AG (7-nt deletion downstream of the PBS motif and G/C/A insertion at the 3′ end of fragment 2). These switchable conformations contain the established structural elements essential for HIV-1 reverse transcription initiation as well as several elements that may also be relevant to initiation process, namely hairpins with GAAA apical loops and self-contained motifs of the duplicate insertion and the downstream palindromic sequence. Taken together, our findings suggest a role for the duplicate insertion of MAL-like templates in HIV-1 reverse transcription initiation process and possible mechanisms to realize this role.

Structure, assembly and dynamics of macromolecular complexes by single particle cryo-electron microscopy

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Multivalent Engagement of TFIID to Nucleosomes

The process of eukaryotic transcription initiation involves the assembly of basal transcription factor complexes on the gene promoter. The recruitment of TFIID is an early and important step in this process. Gene promoters contain distinct DNA sequence elements and are marked by the presence of post-translationally modified nucleosomes. The contributions of these individual features for TFIID recruitment remain to be elucidated. Here, we use immobilized reconstituted promoter nucleosomes, conventional biochemistry and quantitative mass spectrometry to investigate the influence of distinct histone modifications and functional DNA-elements on the binding of TFIID. Our data reveal synergistic effects of H3K4me3, H3K14ac and a TATA box sequence on TFIID binding in vitro. Stoichiometry analyses of affinity purified human TFIID identified the presence of a stable dimeric core. Several peripheral TAFs, including those interacting with distinct promoter features, are substoichiometric yet present in substantial amounts. Finally, we find that the TAF3 subunit of TFIID binds to poised promoters in an H3K4me3-dependent manner. Moreover, the PHD-finger of TAF3 is important for rapid induction of target genes. Thus, fine-tuning of TFIID engagement on promoters is driven by synergistic contacts with both DNA-elements and histone modifications, eventually resulting in a high affinity interaction and activation of transcription.

Effects of Rate-Limiting Steps in Transcription Initiation on Genetic Filter Motifs

The behavior of genetic motifs is determined not only by the gene-gene interactions, but also by the expression patterns of the constituent genes. Live single-molecule measurements have provided evidence that transcription initiation is a sequential process, whose kinetics plays a key role in the dynamics of mRNA and protein numbers. The extent to which it affects the behavior of cellular motifs is unknown. Here, we examine how the kinetics of transcription initiation affects the behavior of motifs performing filtering in amplitude and frequency domain. We find that the performance of each filter is degraded as transcript levels are lowered. This effect can be reduced by having a transcription process with more steps. In addition, we show that the kinetics of the stepwise transcription initiation process affects features such as filter cutoffs. These results constitute an assessment of the range of behaviors of genetic motifs as a function of the kinetics of transcription initiation, and thus will aid in tuning of synthetic motifs to attain specific characteristics without affecting their protein products.

Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

BackgroundInitiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.ResultsBased on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.ConclusionAs demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners with high precision and accuracy.