Jack Horowitz

Isolation and partial characterization of Escherichia coli valine transfer RNA with uridine and uridine-derived residues replaced by 5-fluorouridine☆

Abstract To examine the role of minor bases in transfer RNA, valine 1 transfer RNA of Escherichia coli with the majority of its uridine and uridine-derived residues replaced by fluorouridine, was purified by benzoylated DEAE-cellulose chromatography. 87% replacement of pseudouridine, 95% replacement of ribothymidine, dihydrouridine, 4-thiouridine, and uridine and 74% replacement of the uridine-5-oxyacetic acid were achieved. 6% of the cytidine was replaced by 5-fluorocytidine. Comparison of the ultraviolet absorbance—temperature profiles and circular dichroism spectra of normal and analogue-containing tRNA indicated that this extensive base substitution had no major effects on the secondary structure of the molecule. The minor bases do not appear to be essential for proper recognition by synthetase since in the aminoacylation reaction with partially purified valyl-tRNA synthetase, normal and 5-fluorouridine-containing valine tRNA had virtually the same K m , 1.5 to 1.6 × 10 −7 m .

Characterization of ribosomes and RNAs from Mycoplasma hominis.

Abstract 1. The RNAs and ribosomes of Mycoplasma hominis have been characterized as part of a study to determine whether the protein synthesizing apparatus of an organism with a limited genome is significantly less complex than that of more extensively studied prokaryotes such as Escherichia coli . 2. Ribosomes from M. hominis sediment at 71 S and are composed of 61 % RNA and 39 % protein. These ribosomes dissociate into subunits with sedimentation constants of 33 S and 53 S in buffers containing 0.5–1.0 mM Mg 2+ . 3. The two species of high molecular weight RNA isolated from mycoplasma have sedimentation coefficients of 22 S and 16 S; the larger component sedimenting slightly slower than the 23-S rRNA of E. coli . 4. A species of RNA equal in size to E. coli 5-S RNA has been isolated from M. hominis by gel filtration. It differed from E. coli 5-S RNA in base composition and chromatographic behavior on methylated albumin-kieselguhr columns. The molar ratio of this RNA to high molecular weight rRNA was approximately 0.9 : 1. 5. Transfer RNA from mycoplasma has sedimentation and gel filtration properties identical to E. coli tRNA and can be charged with amino acids by the aminoacyl-tRNA ligases of E. coli . Analysis of the minor base composition indicates that M. hominis tRNA contains about half the amount of pseudouridine, dihydrouridine, and 4-thiouridine and less than 1 % of the ribothymidine found in E. coli tRNA.

Protein synthetic ability of Escherichia coli valine transfer RNA with pseudouridine, ribothymidine, and other uridine-derived residues replaced by 5-fluorouridine

Abstract The requirement for pseudouridine and other uridine-derived minor nucleotides for activity of transfer RNA in several of the intermediate steps in protein synthesis was examined using a purified preparation of Escherichia coli valine transfer RNA in which the uridine and uridine-derived nucleotides were replaced by 5-fluorouridine. The degree of substitution was 87% or better for uridine, pseudouridine, ribothymidine, dihydrouridine, and 4-thiouridine, and at least 75% for uridine-5-oxyacetic acid. Each of these nucleotides, except for uridine, occurs only once in this transfer RNA species. The rate and yield of ternary complex formation with elongation factor Tu-GTP of E. coli , the rate and extent of elongation factor-dependent binding to ribosomes at 10 m m -Mg 2+ , and the rate and extent of synthesis of the co-polypeptide (Phe n ,Val) dependent on poly(U 3 ,G) were all unchanged when the fluorouridine-containing transfer RNA was used in place of the normal control. In all yield assays, the amount of product formed was proportional to the amount of valyl-tRNA added. Non-enzymatic binding to ribosomes in the presence of tetracycline was more efficient for the fluorouridine-substituted tRNA than for the control. At 15 to 20 m m -Mg 2+ the polynucleotide-dependent binding, as a percentage of tRNA added, was 44% for the control and 65% for the modified tRNA, while at 5 m m -Mg 2+ , the figures were 10% and 40%, respectively. We conclude from these results that there is no essential requirement for pseudouridine or ribothymidine in the GTψC loop of tRNA for its proper functioning in protein synthesis in vitro . Confirming earlier work, dihydrouridine and 4-thiouridine are also not essential.

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3 end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5 end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.

Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu.

Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis. Valyl-tRNA(Val) terminating in 3-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3 end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3 cytosine. Similar observations were made in studies of the interaction of 3 end mutants of E. coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3 end of aminoacyl-tRNAs.

Affinity binding of Escherichia coli ribosomal proteins to immobilized RNA

The ribosome consists of a complex array of proteins and RNA arranged in a specific three-dimensional structure. Interactions between ribosomal proteins and ribosomal RNA are essential for the maintenance of this structure in an active conformation [ I] . As part of a study of the recognition process between ribosomal proteins and RNA molecules, we have explored the use of affinity chromatography as a means of isolating and identifying the ribosomal proteins that form specific complexes with RNA or polynucleotide fragments. In this preliminary communication, we report an improved method, based on the nucleotide coupling procedure of Lamed et al. [2], for covalently linking RNA molecules, via their 3’-terminus, to an agarose gel and, as an example of the utility of this material, describe its use to probe the interactions between matrix-bound 5s RNA and E. coli ribosomal proteins. In addition, we have made a simple and inexpensive modification of the basic Kaltschmidt and Wittmann two-dimensional gel electrophoresis apparatus [3] , which permits the use of reduced-size gel slabs.

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA☆

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.

Formation of active β-galactosidase by Escherichia coli treated with 5-fluorouracil☆

Abstract Inhibition by 5-fluorouracil of β-galactosidase synthesis in Escherichia coli has been studied. The analogue prevents formation of active β-galactosidase by cells growing in a medium containing a readily catabolizable carbon source such as glycerol. However, extensive synthesis of enzyme in the presence of the fluoropyrimidine can be observed under a number of conditions known to relieve the effects of catabolite repression. These include removal of the carbon source, glycerol; substitution of other sources of energy for glycerol, most notably succinate; and a shift from aerobic to anaerobic growth conditions. In succinate medium the 5-fluorouracil-treated cells synthesize β-galactosidase at a differential rate more than half that in the absence of analogue. The fluoropyrimidine is readily incorporated into cells in this medium and is found to sediment with the messenger RNA (mRNA) fraction on sucrose gradients. These results suggest that functional mRNA specific for β-galactosidase can be synthesized in the presence of 5-fluorouracil.

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA☆

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA: A 19F nuclear magnetic resonance relaxation study

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the two-state jump and diffusion in a cone formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).