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J Ofengand

Publications and source records attributed to J Ofengand.

At least 91 records · Page 5Linked to original sources
PubMed1979

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.

Ascorbic Acid↗
PubMed1979

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.

Dinitrophenols↗
PubMed1978

Replacement of pseudouridine in transfer RNA by 5-fluorouridine does not affect the ability to stimulate the synthesis of guanosine 5'-triphosphate 3'-diphosphate.

The requirement for ribothymidine and pseudouridine in the TpsiCG loop of tRNA for its activity in the ribosome and tRNA-stimulated synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) by stringent factor has been tested by the use of a purified tRNAPhe (883 pmol of phenylalanine incorporated/A260 unit) in which 92% of the pseudouridine, 98% of the ribothymidine, 98% of the dihydrouridine, and 88% of the uridines were substituted by 5-fluorouridine. This tRNA was quantitatively as active as control tRNA in inducing pppGpp synthesis. With loose-couple ribosomes, the concentration of tRNA needed to give half-maximal reaction was 0.07 micrometer for both normal and fluorouridine-substituted tRNA, with vacant tight-couple ribosomes it was 0.05 micrometer, and with tight couples carrying poly(Phe)-tRNA at the P site the value was 0.02 micrometer. These results show that at the level of intact tRNA there is no special requirement for modified bases in the TpsiCG loop of tRNA in the synthesis of pppGpp.

Escherichia coli↗
PubMed1978

Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site.

Acetylvalyl-, acetylphenylalanyl-, and formylmethionyl-tRNA which were derivatized at their 4-thiouridine residues with the photoaffinity label, p-azidophenacyl bromide, were nonenzymatically bound to salt-washed ribosomes. More than 90% of the binding was to the P site as judged by reactivity with puromycin. Subsequent irradiation (greater than 310 nm) of the tRNA-ribosome complexes resulted in the covalent linking of only the acetylvalyl-tRNA to the 30S subunit. Attachment was solely to the 16S RNA with an efficiency of cross-linking of 13--15%. Covalent linking was 90% inhibited by prior treatment with puromycin, showing that the covalent linking reaction had taken place at the P site. Cross-linking required irradiation and mRNA but was not dependent on the presence of the photoaffinity probe in the tRNA. tRNAs whose 4-thiouridine had been modified with unreactive analogues of p-azidophenacyl bromide or unmodified acetylvalyl-tRNA exhibited the same cross-linking behavior as photoaffinity probe-modified acetylvalyl-tRNA. Furthermore, even acetylvalyl-tRNA whose 4-thiouridine had been removed by treatment with H2O2 was quantitatively as active as unmodified tRNA. These results provide the first demonstration of direct photochemical cross-linking of tRNA to ribosomes.

Affinity Labels↗
PubMed1978

Ability of modified forms of phenylalanine tRNA to stimulate guanosine pentaphosphate synthesis by the stringent factor-ribosome complex of E. coli.

tRNA(Phe) of E. coli, modified at its 4-thiouridine ((4)Srd) and 3-(3-amino-3-carboxypropyl)uridine (nbt(3)Urd) residues, was tested for its ability to induce (p)ppGpp synthesis. The (4)Srd residue was derivatized with the p-azido-phenacyl group, cross-linked to Cyd(13), and the borohydride reduction product of the cross-link was prepared. The nbt(3)Urd residue was derivatized with the N-(4-azido-2-nitrophenyl)glycyl group. None of these derivatives had more than a minor effect on the affinity of the tRNA for the stringent factor-ribosome complex, and no effect at all on the maximum velocity of (p)ppGpp synthesis, either at 2 or 82 mM NH(4)Cl. These two regions of the tRNA which are on opposite faces of the tRNA molecule do not appear to be structurally important for recognition by the stringent factor-ribosome complex. They may provide useful sites, therefore, for the introduction of photoaffinity or fluorescent probes with which to study tRNA-stringent factor recognition.

Ammonium Chloride↗
PubMed1977

Synthesis and properties of nucleoside 5'-phosphoazidates derived from guanosine and adenosine nucleotides: effective on elongation factors G and Tu dependent reactions.

A new type of nucleoside poly (5'-phosphate) analogue, nucleoside 5"-phosphoazidate, with an azido group on the terminal phosphate of GTP, ATP, GDP, GMP, AND AMP, has been synthesized by nucleophilic displacement of the corresponding activated nucleosides by azide ion in yields of 25-45%. Guanosine 5-phosphoazidates is readily photolyzed by ultraviolet light; the corresponding adenosine derivative photolyzes more slowly. Guanosine 5'-O-(2-azidodiphosphate) and guanosine 5"-O-(3-azidotrophosphate) are competitive inhibitors of the formation of the ribosome-EF-G-GDP-fusidic acid complex and of the ribosome-EF-G GTPase. The dissociation constants of the former reaction are calculated to be 27 and 7 micrometer, respectively, or 270 and 73 times that of GDP. In the latter reaction, which is conducted in the absence of fusidic acid, the Ki values are 330 and 28 micrometer, respectively, or 12 and 1 times that of GDP. Guanosine 5"-O-(2-azidodiphosphate) and guanosine 5'-O-(3-azidotriphosphate) also complete with GTP in the formation of the binary complex. EF-Tu-GTP, with respective Kd values of 750 and 75 relative to GTP.

Adenine Nucleotides↗
PubMed1977

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.

Escherichia coli↗
PubMed1975

Photoaffinity labeling of the ribosomal A site with S-(p-azidophenacyl)valyl-tRNA.

S-(p-Azidophenacyl)valyl-tRNA, an analog of valyl-tRNA which has a photoaffinity label attached to its 4-thiouridine residue, was bound to the ribosomal A site at 10 mM Mg2+. Binding was stimulated 25-fold by the presence of elongation factor EFTu. Photoactivation of the p-azidophenacyl group by irradiation resulted in covalent linking of 6% of the noncovalently bound tRNA to the ribosomes. Covalent linking was dependent on the simultaneous presence of ribosomes, poly(U2,G),EFTu.GTP, required irradiation, and did not occur when S-(phenacyl)valyl-tRNA, a nonphotolyzable analog, replaced S-(p-azidophenacyl)valyl-tRNA. The attached tRNA was distributed approximately equally between both the 30S and 50S subunits. At the 30S subunit, 30% of the tRNA was bound to protein while 70% was linked to 16S RNA. At the 50S subunit, however, negligible binding to the 23S RNA was observed. More than 90% of the tRNA was attached to low molecular weight material according to sodium dodecyl sulfate-sucrose gradient analysis, and more than 87% of this fraction consisted of tRNA-protein complexes as assayed by phenol solubility and electrophoretic mobility before and after protease treatment. These results, in conjunction with our previous report (I. Schwartz and J Ofengand (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 3951) which showed that covalent linking of this same tRNA derivative at the ribosomal P site resulted in attachment solely to the 16S RNA, demonstrate that 16S, but not 23S or 5S rRNA, is an important component of the tRNA binding site in the region of the 4-thiouridine residue and furthermore show that ribosomal A and P sites are topologically distinct.

Affinity Labels↗
PubMed1974

Photo-affinity labeling of tRNA binding sites in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyl-tRNA to 16S RNA at the ribosomal P site.

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA(1) (Val) was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[(3)H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 A of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.

Binding Sites↗
PubMed1974

Terminal oxidation-reduction of N-acetyl-phenylalanyl-tRNA blocks initiation complex formation with Escherichia coli 30S ribosomal subunits.

Terminally oxidized-reduced tRNA(Phe) of yeast, exclusively acylated at the 2'-hydroxyl of the 3'-terminal ribose of the tRNA, is a useful model for investigating the stereospecificity of AA-tRNA in protein synthesis. In this work, the ability of N-acetyl-Phe-tRNA(ox-red) to form an initiation complex with Escherichiacoli 30S ribosomal subunits was investigated. Thirty per cent of added control N-acetyl-Phe-tRNA was bound in a reaction dependent on initiation factors, GTP, and poly U, but no binding of the oxidized-reduced analog could be detected. These results imply that initiation complex formation may be specific for the 3'-ester of initiator tRNA.

Binding Sites↗