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At least 415 records · Page 23Linked to original sources
PubMed2006

The NUG1 GTPase reveals and N-terminal RNA-binding domain that is essential for association with 60 S pre-ribosomal particles.

The putative yeast GTPase Nug1, which is associated with several pre-60 S particles in the nucleolus and nucleoplasm, consists of an N-terminal domain, which is found only in eukaryotic orthologues, and middle and C-terminal domains that are conserved throughout eukaryotes, bacteria, and archaea. Here, we analyzed the role of the eukaryote-specific Nug1 N-domain (Nug1-N). We show that the essential Nug1-N is sufficient and necessary for nucle(ol)ar targeting and association with pre-60 S particles. Nug1-N exhibits RNA binding activity and is genetically linked in an allele-specific way to the pre-60 S factors Noc2, Noc3, and Dbp10. In contrast, the middle domain, which exhibits a circularly permuted GTPase fold and an intrinsic GTP hydrolysis activity in vitro, is not essential for cell growth. The conserved Nug1 C-domain, which has a yet uncharacterized fold, is also essential for ribosome biogenesis. Our findings suggest that Nug1 associates with pre-60 S subunits via its essential N-terminal RNA-binding domain and exerts a non-essential regulative role in pre-60 S subunit biogenesis via its central GTPase domain.

Binding Sites↗
PubMed2005

Relevance of nuclear localization and functions of RNA helicase A.

RNA helicase A (RHA) regulates gene expression at transcriptional and post-transcriptional levels. It is also known that RHA shuttles between the nucleus and the cytoplasm. RHA possesses nuclear transport domain which functions as a nuclear localization signal (NLS) and nuclear export signal (NES), and predominantly localizes in the nucleus. Here, we identified regions that influence cellular localization of RHA. We showed that double-stranded RNA binding domain I and II (dsRBD I and II) positively localized to the cytoplasm. Mutational analysis revealed that RNA-binding ability was important to localization of dsRBD to the cytoplasm. RHA mutants lacking association with Pol II complexes were partially spilled from the nucleus. Furthermore, a minimal transactivation domain (MTAD), which is minimal region to interact with Pol II complexes, mainly localized to the nucleus in comparison with GFP. Our results indicated that the intracellular localization of RHA might be regulated by not only NLS and NES but also functional domains.

Autoantigens↗
PubMed2002

Nuclear export and retention signals in the RS domain of SR proteins.

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.

3T3 Cells↗
PubMedDate not supplied

Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.

The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 A, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor-acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids.

Base Sequence↗
PubMed2006

Interplay between the beta' clamp and the beta' jaw domains during DNA opening by the bacterial RNA polymerase at sigma54-dependent promoters.

The bacterial RNA polymerase (RNAP) is a multi-subunit, structurally flexible, complex molecular machine, in which activities associated with DNA opening for transcription-competent open promoter complex (OC) formation reside in the catalytic beta and beta' subunits and the dissociable sigma subunit. OC formation is a multi-step process that involves several structurally conserved mobile modules of beta, beta', and sigma. Here, we present evidence that two flexible modules of beta', the beta' jaw and the beta' clamp and a conserved regulatory Region I domain of sigma(54), jointly contribute to the maintenance of stable DNA strand separation around the trancription start site in OCs formed at sigma(54)-dependent promoters. Clearly, regulated interplay between the mobile modules of the beta' and the sigma subunits of the RNAP appears to be necessary for stable OC formation.

DNA↗
PubMed2001

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7.

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-beta family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-beta related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.

Animals↗
PubMed2003

Bacterial RNA polymerases: the wholo story.

Recent structural and biophysical results have provided unprecedented insights into the structure and function of the bacterial RNA polymerase holoenzyme as it goes through the steps of transcription initiation. Comparisons with structural analyses of evolutionarily unrelated RNA polymerases reveal unexpected general features of the initiation process.

Bacteria↗
PubMed2001

An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA.

The crystal structure of Thermotoga maritima NusA, a transcription factor involved in pausing, termination, and antitermination processes, reveals a four-domain, rod-shaped molecule. An N-terminal alpha/beta portion, a five-stranded beta-barrel (S1 domain), and two K-homology (KH) modules create a continuous spine of positive electrostatic potential, suitable for nonspecific mRNA attraction. Homology models suggest how, in addition, specific mRNA regulatory sequences can be recognized by the S1 and KH motifs. An arrangement of multiple S1 and KH domains mediated by highly conserved residues is seen, creating an extended RNA binding surface, a paradigm for other proteins with similar domain arrays. Structural and mutational analyses indicate that the motifs cooperate, modulating strength and specificity of RNA binding.

Bacterial Proteins↗
PubMed2002

VASA localization requires the SPRY-domain and SOCS-box containing protein, GUSTAVUS.

VASA (VAS), a key protein in establishing the specialized translational activity of the Drosophila pole plasm, accumulates at the posterior pole of the developing oocyte. We identified a gene, gustavus (gus), that encodes a protein that interacts with VAS. A gus mutation blocks posterior localization of VAS, as does deletion of a segment of VAS containing the GUS binding site. Like VAS, GUS is present in cytoplasmic ribonucleoprotein particles. Heterozygotes for gus or a deletion including gus produce embryos with fewer pole cells and posterior patterning defects. Therefore, GUS is essential for the posterior localization of VAS. However, gus is not required for the posterior localization of oskar (osk). Apparent gus orthologs are present in mammalian genomes.

Animals↗
PubMed2002

Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.

The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.

Amino Acid Sequence↗
PubMed2001

Residues 137 and 153 at the N terminus of the XylS protein influence the effector profile of this transcriptional regulator and the sigma factor used by RNA polymerase to stimulate transcription from its cognate promoter.

The 321-residue XylS and XylS1 proteins, encoded by the pWW0 and pWW53 plasmids respectively, differ in only 5 residues at positions 4, 53, 90, 137, and 153. As a result, the effector profile of XylS is wider than that of XylS1, and XylS mediates higher levels of transcription from its cognate-regulatable promoter than does XylS1. We generated a series of XylS-pWW0 mutants and found that the single mutants Asp-137-->Glu and His-153-->Asn exhibited an activation pattern different from that of the wild-type regulator. In the double-mutant XylSD137E,H153N the effector profile for benzoates was similar to that of XylS1. This suggests that these two residues are crucial for effector recognition and regulator activation to stimulate transcription. XylS-dependent transcription from its cognate promoter is mediated by RNA polymerase with sigma(32) or sigma(38), whereas XylS1 uses RNA polymerase with sigma(32) or sigma(70). We also found that point mutations at positions 137 and 153 of XylS led RNA polymerase to mediate transcription with sigma(70) rather than with sigma(38), as demonstrated by primer extension analysis in a sigma(70)-thermosensitive background proficient and deficient in sigma(38). This suggests that a positive transcriptional regulator can choose the RNA polymerase complex that mediates transcription from a given promoter.

Amino Acids↗
PubMed2006

Association of RNA helicase a with human immunodeficiency virus type 1 particles.

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription.

Autoantigens↗
PubMed2004

Human LZIP binds to CCR1 and differentially affects the chemotactic activities of CCR1-dependent chemokines.

Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1-mediated cellular signal transduction mechanism, we used the yeast two-hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two-hybrid assay and immunoprecipitation experiments. The 21-260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn-1-induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP-1alpha, RANTES, or HCC-4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.

Animals↗
PubMed2005

Function of the conserved S1 and KH domains in polynucleotide phosphorylase.

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.

Binding Sites↗
PubMed2003

Functional characterization of the mammalian mRNA decapping enzyme hDcp2.

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.

Amino Acid Motifs↗
PubMed2006

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold.

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.

Amino Acid Motifs↗
PubMed2005

RNA-binding domain proteins in Kinetoplastids: a comparative analysis.

RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

Amino Acid Motifs↗