Single RNA Polymerase Binding Site Isolated

One approach to study the structure of promoter regions^1–3 is to isolate RNA polymerase binding sites. Several attempts have been made to isolate such sites as protected DNA fragments^4–10, but so far the DNA isolated has not definitely shown to be only one or a few specific sites. We report here the isolation of a single RNA polymerase binding site from the replicative form (RF) DNA of bacteriophage fd. The site as isolated is a short double-stranded DNA with a unique nucleotide sequence.     

ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus.

The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T. A portion of this protein (amino acids [aa] 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity. In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro. A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity. The protein also possessed nonspecific RNA binding capacity. Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding. The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.     

Interaction of R17 coat protein with its RNA binding site for translational repression

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kd at 2 degrees C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of Ka and a delta H = -19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RNAs failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific. We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.     

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.     

The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.     

Domain arrangement of Der,a switch protein containing two GTPase domains

The EngA subfamily of essential bacterial GTPases has a unique domain structure consisting of two adjacent GTPase domains (GD1 and GD2) and a C-terminal domain. The structure of Thermotoga maritima Der bound to GDP determined at 1.9 A resolution reveals a novel domain arrangement in which the GTPase domains pack at either side of the C-terminal domain. Unexpectedly, the C-terminal domain resembles a KH domain, missing the distinctive RNA recognition elements. Conserved motifs of the nucleotide binding site of GD1 are integral parts of the GD1-KH domain interface, suggesting the interactions between these two domains are directly influenced by the GTP/GDP cycling of the protein. In contrast, the GD2-KH domain interface is distal to the GDP binding site of GD2.     

Explicit analytic approximations for time-dependent solutions of the generalized integrated Michaelis-Menten equation

A single nucleotide polymorphism (SNP) is a common genetic variation when a single nucleotide differs between members of a species or paired chromosome. Due to its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we present two quantitative SNP detection methods for a single-base mismatch in RNA, based on nick-joining and nick-generating activities of T4 RNA ligase and DNAzyme, respectively. T4 RNA ligase successfully discriminated a one-base mismatch in the ligation junction, and the designed DNAzyme cleaved RNA by discerning a single-base mismatch in the cleaving site.     

Interaction of R17 Coat Protein With Its RNA Binding Site For Translational Repression

Abstract

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kj at 2°C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of K_a and a ΔH = ?19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RN As failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific.

We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.     

Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J

RNase J is a key member of the β-CASP family of metallo-β-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B.?subtilis enzyme possesses both 5'-3' exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T.?thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5'-3' exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5'-3' exonucleolytic mode. We show that B.?subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the β-CASP domain to explain the enzyme's processive action.