Disordered nucleiome: Abundance of intrinsic disorder in the DNA‐ and RNA‐binding proteins in 1121 species from Eukaryota,Bacteria and Archaea

Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty‐by‐association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (～548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA‐binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA‐binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation.     

固有无序蛋白质及逆境胁迫下的分子功能

固有无序蛋白质是一类在生理条件下缺乏稳定三维结构而具有正常功能,参与信号转导、转录调控、胁迫应答等多种生物学过程的蛋白质.植物中许多逆境响应蛋白是固有无序蛋白质,通过其结构无序或部分无序区域在蛋白质 蛋白质、蛋白质 膜脂、蛋白质 核酸的互作中发挥重要作用.本文主要对固有无序蛋白质的类别、氨基酸组成和结构特点以及在逆境胁迫下其稳定细胞膜、保护核酸和蛋白质、调控基因表达等分子功能进行综述,以拓展对逆境胁迫下蛋白质作用分子机制的认识.     

p53-catalyzed annealing of complementary single-stranded nucleic acids.

p53 has been reported to inhibit the DNA helicase intrinsic to simian virus 40 large tumor antigen (T antigen). We found that inhibition is not restricted to T antigen, but also affects several other DNA and RNA helicases. Complexing of the helicases by the p53 protein as a possible inactivation mechanism could be excluded. Instead, the anti-helicase activity can be explained by our finding that p53 binds with high affinity to single-stranded nucleic acids and has a strong DNA.DNA and RNA.RNA annealing activity. We could also show that p53 is able to alter the secondary structure of RNA and/or to influence dynamic RNA-RNA interactions. These results, and the fact that the affinity of p53 to RNA is about one order of magnitude higher than to single-stranded DNA, imply an RNA-specific function of p53 in vivo.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

Mechanisms of DNA binding determined in optical tweezers experiments

The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments.     

Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function

Glycine-rich RNA-binding proteins (GR-RBPs) are involved in cold shock response of plants as RNA chaperones facilitating mRNA transport, splicing and translation. GR-RBPs are bipartite proteins containing a RNA recognition motif (RRM) followed by a glycine-rich region. Here, we studied the structural basis of nucleic acid binding of full-length Nicotiana tabacum GR-RBP1. NMR studies of NtGR-RBP1 show that the glycine-rich domain, while intrinsically disordered, is responsible for mediating self-association by transient interactions with its RRM domain (NtRRM). Both NtGR-RBP1 and NtRRM bind specifically and with low micromolar affinity to RNA and single-stranded DNA. The solution structure of NtRRM shows that it is a canonical RRM domain. A HADDOCK model of the NtRRM–RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues. Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM. Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR.

trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA. Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined. Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo. Rather, the activation domain is also required and must be juxtaposed to the basic domain. Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.     

Classifying RNA-binding proteins based on electrostatic properties

Protein structure can provide new insight into the biological function of a protein and can enable the design of better experiments to learn its biological roles. Moreover, deciphering the interactions of a protein with other molecules can contribute to the understanding of the protein's function within cellular processes. In this study, we apply a machine learning approach for classifying RNA-binding proteins based on their three-dimensional structures. The method is based on characterizing unique properties of electrostatic patches on the protein surface. Using an ensemble of general protein features and specific properties extracted from the electrostatic patches, we have trained a support vector machine (SVM) to distinguish RNA-binding proteins from other positively charged proteins that do not bind nucleic acids. Specifically, the method was applied on proteins possessing the RNA recognition motif (RRM) and successfully classified RNA-binding proteins from RRM domains involved in protein-protein interactions. Overall the method achieves 88% accuracy in classifying RNA-binding proteins, yet it cannot distinguish RNA from DNA binding proteins. Nevertheless, by applying a multiclass SVM approach we were able to classify the RNA-binding proteins based on their RNA targets, specifically, whether they bind a ribosomal RNA (rRNA), a transfer RNA (tRNA), or messenger RNA (mRNA). Finally, we present here an innovative approach that does not rely on sequence or structural homology and could be applied to identify novel RNA-binding proteins with unique folds and/or binding motifs.     

Prion Propagation: The Role of Protein Dynamics

The transfer of phenotypes from one individual to another is a fundamental aspect of biology. In addition to traditional nucleic acid-based genetic determinants, unique proteins known as prions can also act as elements of inheritance, infectivity, and disease. Nucleic acids and proteins encode genetic information in distinct ways, either in the sequence of bases in DNA or RNA or in the three dimensional structure of the polypeptide chain. Given these differences in the nature of the genetic repository, the mechanisms underlying the transmission of nucleic acid-based and protein-based phenotypes are necessarily distinct. While the appearance, persistence and transfer of nucleic acid determinants require the synthesis of new polymers, recent studies indicate that prions are propagated through dynamic transitions in the structure of existing protein.     

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ~10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Nucleoporins’ exclusive amino acid sequence features regulate their transient interaction with and selectivity of cargo complexes in the nuclear pore

Nucleocytoplasmic traffic of nucleic acids and proteins across the nuclear envelop via the nuclear pore complexes (NPCs) is vital for eukaryotic cells. NPCs screen transported macromolecules based on their morphology and surface chemistry. This selective nature of the NPC-mediated traffic is essential for regulating the fundamental functions of the nucleus, such as gene regulation, protein synthesis, and mechanotransduction. Despite the fundamental role of the NPC in cell and nuclear biology, the detailed mechanisms underlying how the NPC works have remained largely unknown. The critical components of NPCs enabling their selective barrier function are the natively unfolded phenylalanine- and glycine-rich proteins called “FG-nucleoporins” (FG Nups). These intrinsically disordered proteins are tethered to the inner wall of the NPC, and together form a highly dynamic polymeric meshwork whose physicochemical conformation has been the subject of intense debate. We observed that specific sequence features (called largest positive like-charge regions, or lpLCRs), characterized by extended subsequences that only possess positively charged amino acids, significantly affect the conformation of FG Nups inside the NPC. Here we investigate how the presence of lpLCRs affects the interactions between FG Nups and their interactions with the cargo complex. We combine coarse-grained molecular dynamics simulations with time-resolved force distribution analysis to disordered proteins to explore the behavior of the system. Our results suggest that the number of charged residues in the lpLCR domain directly governs the average distance between Phe residues and the intensity of interaction between them. As a result, the number of charged residues within lpLCR determines the balance between the hydrophobic interaction and the electrostatic repulsion and governs how dense and disordered the hydrophobic network formed by FG Nups is. Moreover, changing the number of charged residues in an lpLCR domain can interfere with ultrafast and transient interactions between FG Nups and the cargo complex.     

Refinement of the structure of protein–RNA complexes by residual dipolar coupling analysis

The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.     

Nucleic acid aptamers and enzymes as sensors

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.     

Evaluation of single-stranded nucleic acids as carriers in the DNA-directed assembly of macromolecules

Current developments in nanosciences indicate that the self-assembly of macromolecules, such as proteins or metallic nanoclusters, can be conveniently achieved by means of nucleic acid hybridization. Within this context, we here report on the evaluation of single-stranded nucleic acids to be utilized as carrier backbones in DNA-directed self-assembly. A microplate solid-phase hybridization assay is described which allows rapid experimental determination of the hybridization efficiencies of various sequence stretches within a given nucleic acid carrier strand. As demonstrated for two DNA fragments of different sequence, the binding efficiencies of several oligonucleotides depend on the formation of specific secondary structure elements within the carrier molecule. A correlation of sequence-specific hybridization capability with modeled secondary structure is also obvious from experiments using the fluorescence gel-shift analysis. Electrophoretic studies on the employment of helper oligonucleotides in the formation of supramolecular conjugates of several oligonucleotide-tagged proteins indicate, that structural constraints can be minimized by disruption of intramolecular secondary structures of the carrier molecule. To estimate the influences of the chemical nature of the carrier, gel-shift experiments are carried out to compare a 170mer RNA molecule with its DNA analogue. Ternary aggregates, containing two protein components bound to the carrier, are formed with a greater efficiency on the DNA instead of the RNA carrier backbone.     

Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.     

Lysozyme association with nucleic acids

Lysozyme is well known for the ability to hydrolyze the cell wall of bacteria. Based on the similarity of structure between lysozyme and histones as seen from the results of X-ray crystal structure determinations, we have postulated that binding to nucleic acids may be another biological function of lysozyme. We have therefore begun a systematic study of the interactions of lysozyme and related molecules with nucleic acids, and present here a preliminary report. Binding to DNA and RNA has been demonstrated from gel electrophoresis, enzyme activity, and coprecipitation studies. We suggest that this function of lysozyme will provide an explanation why Lee-Huang et al. (1999) [Proc. Natl. Acad. Sci. USA 96, 2678-2681] were able to call lysozyme a "killer protein" against the AIDS virus, and may provide a new avenue of research on AIDS therapy.