Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.     

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3'' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.     

3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U)

The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3′ nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3′ X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3′ NTR, 3′ SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3′ SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3′ X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.     

Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.     

Enrichment of a second class of native acceptor sites for the avian oviduct progesterone receptor as intact chromatin fragments

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing approximately 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA.

Poly(A) site processing of a pre-mRNA requires the participation of multiple nuclear factors. Two of these factors recognize specific sequences in the pre-mRNA and form a stable processing complex. Since these initial interactions are likely critical for the recognition of the poly(A) site and the efficiency of poly(A) site use, we have characterized these factors and the nature of their interaction with the pre-mRNA. The AAUAAA specificity factor PF2 is a large, multicomponent complex composed of at least five distinct polypeptides ranging in molecular size from 170 to 42 kDa. The 170-kDa polypeptide appears to mediate interaction with the pre-mRNA. Factor CF1, which provides specificity for the downstream G + U-rich element and stabilizes the PF2 interaction on the RNA, is also a multicomponent complex but is less complex than PF2. CF1 is composed of three polypeptides of molecular sizes 76, 64, and 48 kDa. UV cross-linking assays demonstrate that the 64-kDa polypeptide makes direct contact with the RNA, dependent on the G + U-rich downstream sequence element. Moreover, it is clear that these RNA-protein interactions are influenced by the apparent cooperative interaction involving PF2 and CF1, interactions that contribute to the efficiency of poly(A) site processing.     

Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid

When annealed with synthetic polynucleotides and treated with ribonuclease under appropriate conditions, poly(U) forms the ribonuclease-resistant complexes poly(rA) · poly(U) (1:1), poly(dA) · 2poly(U) (1:2) and poly · (dA)poly(dT) · poly(U) (1:1:1). This forms the basis of a quantitative assay of poly(rA), poly(dA) and poly(dA) · poly(dT) sequences in unlabelled nucleic acids. Using this assay, duck haemoglobin messenger RNA is shown to contain a poly(rA) sequence approximately 100 nucleotides long.Eukaryotic DNAs contain small amounts of sequences that react with poly(U). In the case of duck DNA, these sequences are considerably shorter than the mRNA-associated sequences and are interspersed widely with other sequences. It is concluded that if duck DNA does contain poly(dA) sequences corresponding to mRNA-associated poly(rA) sequences, there are fewer than 8000 of these per haploid genome.     

Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein.

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.     

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3′-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1’s activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1’s activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.     

4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells

Monkey kidney cells (CV-1) cultivated in the presence of 0.1 mM 4-thiouridine (S4U) and subsequently illuminated at 365 nm exhibit a marked RNA synthesis inhibition. Maximal effect (approximately 40%) was obtained for a 4 h S4U incubation and a 45 KJ/m2 dose. Under these conditions up to 20% of total cellular RNA is retained at the interphase during phenol-chloroform extraction. The fraction of RNA crosslinked to proteins amounts to 50% of the 3H-uridine labeled RNA synthesized during S4U incorporation and less than 10% for the control samples. This strongly suggests that S4U incorporated within the RNA chains acts as a photoaffinity probe. The data above provide the basis of a method for studying in vivo RNA-protein interactions under non destructive conditions.