Functional compensation of a recognition-defective transfer RNA by a distal base pair substitution.

A single G3:U70 base pair in the acceptor helix is the major determinant of alanine acceptance in alanine transfer RNAs. Transfer of this base pair into other transfer RNAs confers alanine acceptance. A G3:C70 substitution eliminates alanine acceptance in vivo and in vitro. In this work, a population of mutagenized G3:C70 alanine tRNA amber suppressors was subjected to a selection for mutations that compensate for the inactivating G3:C70 substitution. No compensatory mutations located in the acceptor helix were obtained. Instead, a U27:U43 substitution that replaced the wild-type C27:G43 in the anticodon stem created a U27:U43/G3:C70 mutant alanine tRNA that inserts alanine at amber codons in vivo. The U27:U43 substitution is at a location where previous footprinting work established an RNA-protein contact. Thus, this mutation may act by functionally coupling a distal part of the tRNA structure to the active site.     

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.     

The equilibrium partition function and base pair binding probabilities for RNA secondary structure.

A novel application of dynamic programming to the folding problem for RNA enables one to calculate the full equilibrium partition function for secondary structure and the probabilities of various substructures. In particular, both the partition function and the probabilities of all base pairs are computed by a recursive scheme of polynomial order N3 in the sequence length N. The temperature dependence of the partition function gives information about melting behavior for the secondary structure. The pair binding probabilities, the computation of which depends on the partition function, are visually summarized in a "box matrix" display and this provides a useful tool for examining the full ensemble of probable alternative equilibrium structures. The calculation of this ensemble representation allows a proper application and assessment of the predictive power of the secondary structure method, and yields important information on alternatives and intermediates in addition to local information about base pair opening and slippage. The results are illustrated for representative tRNA, 5S RNA, and self-replicating and self-splicing RNA molecules, and allow a direct comparison with enzymatic structure probes. The effect of changes in the thermodynamic parameters on the equilibrium ensemble provides a further sensitivity check to the predictions.     

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.     

Rapid ribosomal translocation depends on the conserved 18-55 base pair in P-site transfer RNA

The L shape of tRNA is stabilized by the 'tertiary core' region, which contains base-pairing interactions between the D and T loops. Distortions of the L shape accompany tRNA movement across the ribosomal surface. Here, using single-turnover rapid kinetics assays, we determine the effects of mutations within the tertiary core of P site-bound tRNA(fMet) on three measures of the rate of translocation, the part of the elongation cycle involving the most extensive tRNA movement. Mutations in the strictly conserved G18.U55 base pair result in as much as an 80-fold decrease in the rate of translocation, demonstrating the importance of the 18-55 interaction for rapid translocation. This implicates the core region as a locus for functionally important dynamic interactions with the ribosome and leads to the proposal that translocation of ribosome-bound tRNAs may be sequential rather than concerted.     

Simultaneous binding of two proteins to opposite sides of a single transfer RNA

Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase--tRNA--Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery.     

A functional interaction between methionyl-transfer RNA hydrolase and a transfer RNA binding factor

Met-tRNA_f bound at low Mg ion concentrations to rabbit reticulocyte 40 S ribosomal subunits in the presence of ApUpG and a eukaryotic tRNA binding factor serves readily as a substrate for a Met-tRNA hydrolase from rabbit reticulocytes. This hydrolysis occurs rapidly at 0 °C, appears to be specific for Met-tRNA_f, and is not inhibited by 60 S ribosomal subunits. These reactions may be responsible for the accumulation of deacylated tRNA_f^Met observed in ribosomes isolated from sodium fluoride-treated cells.     

Solution structure of an RNA duplex including a C-U base pair

The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.     

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA^Asn and tRNA^Gln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA^Asn and Gln-tRNA^Gln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA^Asn and tRNA^Asp variants, that amidation of Asp acylating tRNA^Asn is promoted by the base pair U₁–A₇₂ whereas the G₁–C₇₂ pair and presence of the supernumerary nucleotide U_20A in the D-loop of tRNA^Asp prevent amidation. We predict, based on comparison of tRNA^Gln and tRNA^Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA^Gln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G₄₆ and U₄₇ of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.     

Thermodynamics of RNA melting,one base pair at a time

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe “microscopic” enthalpy–entropy compensation—often seen “macroscopically” across a series of related molecular species—across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy–entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.     

Synthesis of a dA-dT base pair analogue and its effects on DNA-ligand binding

Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.     

Immunological recognition of the prolactin receptor: identification of a single binding unit of molecular weight approximately 42,000

Different polyclonal and monoclonal antibodies against the rabbit mammary prolactin (PRL) receptor were previously obtained that totally inhibited PRL binding in the rabbit mammary gland. Only polyclonal antibodies were shown to immunoprecipitate preformed PRL--receptor complexes in solubilized mammary membranes suggesting that they also recognized domains outside of the PRL binding site of the receptor. When partially purified PRL receptor preparations from both rabbit and pig mammary tissues were iodinated, immunoprecipitated and subsequently analyzed by SDS--PAGE, a single component of molecular weight approximately 42,000 was specifically recognized by all the anti-PRL receptor antibodies. This unit was the only component immunoprecipitated by the monoclonal antibody M 110. Its identification was not impaired by using reducing or non-reducing conditions. Moreover, a further purification of the [125I]-labeled receptor preparations from both species by a second PRL affinity chromatography selected a single binding unit of the same molecular weight. In contrast, polyclonal antibodies immunoprecipitated additional components apart from the 42,000 unit, especially one unit of molecular weight 70,000-80,000 in both species. We conclude that rabbit and pig mammary PRL receptors exhibit striking immunological similarities. Both contain a single binding unit of molecular weight approximately 42,000 that is not linked to other units via disulfide bridges. This binding unit could be associated with a larger component of MW 70,000-80,000 in the holo receptor.     

A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene

Summary The glyUsu _AGA mutation affects Escherichia coli tRNA _GGG ^{G1 y} , changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base substitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occurring spontaneously has been made also. The frequency of MNNG-induced glyUsu _AGA mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyUsu _AGA mutations, however, strongly suggests that the occurrence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position.In addition to the double change in the anticodon, the glyUsu _AGA tRNA differs from tRNA _GGG ^{G1 y} in that it bears a modification of the A adjacent to the 3 position of the anticodon. Most likely, this modified base is N-[9-(-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine.     

Enzyme specificity: base recognition and hydrolysis of RNA by ribonuclease A

The substrate specificity of pancreatic ribonuclease A is discussed in light of observations based on accurate X-ray structure analysis of several enzyme-nucleotide complexes. A hypothesis for protein-nucleic acid recognition is presented which proposes that: (a) pyrimidine bases in RNA are recognised by ribonuclease due to the charge complementarity of two groups (the amide nitrogen and the side chain oxygen (OG) of threonine 45) of the protein and relevant atoms in the heterocyclic base (O2 and N3 in pyrimidine nucleotides); (b) interaction of the protein with the ribose moiety of the nucleotides is non-specific; and (c) conformational flexibility in the region of the scissile P-O bond is provided by different locations of the phosphoryl oxygens, rather than by an overall translation of the phosphate moiety.     

A single base pair change in proline biosynthesis genes causes osmotic stress tolerance.

A single base pair change has been found in a site corresponding to a regulatory region of the first enzyme in the proline biosynthetic pathway. This change alters feedback inhibition and is responsible for the synthesis of high levels of proline that enable Escherichia coli to withstand osmotic stress.