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J Ofengand

Publications and source records attributed to J Ofengand.

At least 73 records · Page 4Linked to original sources
PubMed1985

Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA.

The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E. coli valyl-tRNA was identified. Cross-linking was via the affinity probe 6-[(2-nitro-4-azidophenyl)amino]caproate (NAK) or 3-[[2-[(2-nitro-4-azidophenyl)amino]ethyl]dithio]propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer [Gornicki, P., Ciesiolka, J., & Ofengand, J. (1985) Biochemistry (preceding paper in this issue)]. With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA. Appearance of the oligomers was dependent on the presence of the probe on the tRNA. Unmodified tRNA in the A and/or P sites did not yield any product. The presence of elongation factor Tu in the incubation mixture was also required. Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers. Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced. The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers. The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length. In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue. The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1985

Conservation of RNA sequence and cross-linking ability in ribosomes from a higher eukaryote: photochemical cross-linking of the anticodon of P site bound tRNA to the penultimate cytidine of the UACACACG sequence in Artemia salina 18S rRNA.

The complex of Artemia salina ribosomes and Escherichia coli acetylvalyl-tRNA could be cross-linked by irradiation with near-UV light. Cross-linking required the presence of the codon GUU, GUA being ineffective. The acetylvalyl group could be released from the cross-linked tRNA by treatment with puromycin, demonstrating that cross-linking had occurred at the P site. This was true both for pGUU- and also for poly(U2,G)-dependent cross-linking. All of the cross-linking was to the 18S rRNA of the small ribosomal subunit. Photolysis of the cross-link at 254 nm occurred with the same kinetics as that for the known cyclobutane dimer between this tRNA and Escherichia coli 16S rRNA. T1 RNase digestion of the cross-linked tRNA yielded an oligonucleotide larger in molecular weight than any from un-cross-linked rRNA or tRNA or from a prephotolyzed complex. Extended electrophoresis showed this material to consist of two oligomers of similar mobility, a faster one-third component and a slower two-thirds component. Each oligomer yielded two components on 254-nm photolysis. The slower band from each was the tRNA T1 oligomer CACCUCCCUVACAAGp, which includes the anticodon. The faster band was the rRNA 9-mer UACACACCGp and its derivative UACACACUG. Unexpectedly, the dephosphorylated and slower moving 9-mer was derived from the faster moving dimer. Deamination of the penultimate C to U is probably due to cyclobutane dimer formation and was evidence for that nucleotide being the site of cross-linking. Direct confirmation of the cross-linking site was obtained by "Z"-gel analysis [Ehresmann, C., & Ofengand, J. (1984) Biochemistry 23, 438-445].(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1984

High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit.

A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E. coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA. By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy. The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid. When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked. Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification. The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented. Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion. Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site.

Antibodies↗
PubMed1984

Cross-linking of the anticodon of Escherichia coli and Bacillus subtilis acetylvalyl-tRNA to the ribosomal P site. Characterization of a unique site in both E. coli 16S and yeast 18S ribosomal RNA.

The nucleotide residues involved in the cross-link between P site bound acetylvalyl-tRNA (AcVal-tRNA) and 16-18S rRNA have been identified. This cross-link was formed by irradiation of Escherichia coli or Bacillus subtilis AcVal-tRNA bound to the P site of E. coli ribosomes or by irradiation of E. coli AcVal-tRNA bound to the P site of yeast ribosomes. The three cross-linked RNA heterodimers were obtained in 10-35% purity by disruption of the irradiated ribosome-tRNA complex with sodium dodecyl sulfate followed by sucrose gradient centrifugation. After total digestion with RNase T1, and labeling at either the 5'- or the 3'-end, the cross-linked oligomers could be identified and isolated before and after photolytic splitting of the cross-link. One of the oligomers was shown to be UACACACCG, a unique rRNA nonamer present in an evolutionarily conserved region. This oligomer was found in all three heterodimers. The other oligomer of the dimer had the sequence expected for the RNase T1 product encompassing the anticodon of the tRNA used. The precise site of cross-linking was determined by two novel methods. Bisulfite modification of the oligonucleotide dimer converted all C residues to U, except for any cross-linked C which would be resistant by being part of a cyclobutane dimer. Sequencing gel analysis of the UACACACCG oligomer showed that the C residue protected was the 3'-penultimate C residue, C1400 in E. coli rRNA or C1626 in yeast rRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1984

Two-dimensional gel electrophoresis technique for determination of the cross-linked nucleotides in cleavable covalent RNA-RNA complexes. Application to Escherichia coli and Bacillus subtilis acetylvalyl-tRNA covalently linked to E. coli 16S and yeast 18S ribosomal RNA.

We have developed a new method which yields in a single step the site of cross-linking between two oligonucleotides covalently linked by a cleavable bond. The isolated duplex, labeled at both 5'-ends, is split randomly and then analyzed by diagonal gel electrophoresis with cleavage of the cross-link between the two gel dimensions. Digestion products which do not contain the cross-link migrate along the diagonal, while products resulting from cleavage of the cross-link migrate as off-diagonal products. The site of cross-linking is determined by analysis of both diagonal and off-diagonal products. This method was successfully applied to three different oligonucleotide duplexes isolated by T1 RNase digestion from Escherichia coli tRNA covalently linked at the ribosomal P site to either Escherichia coli 16S RNA or yeast 18S RNA and from Bacillus subtilis tRNA cross-linked to Escherichia coli 16S RNA. The site of cross-linking was unambiguously localized to C1400 in Escherichia coli 16S RNA and to the equivalent position, C1626, in yeast 18S RNA. Direct evidence was also provided for the participation of the wobble base (c)mo5U34 of the tRNA in the cross-link. Furthermore, our results exclude the possibility of minor cross-linking sites at other positions. This new method is reliable, rapid, and easy to handle and should be applicable to any cleavable covalent RNA-RNA duplex. Furthermore, it is sensitive to certain aspects of the steric conformation of such covalent duplexes.

Bacillus subtilis↗
PubMed1984

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.

Azides↗
PubMed1984

Covalent crosslinking of Escherichia coli phenylalanyl-tRNA and valyl-tRNA to the ribosomal A site via photoaffinity probes attached to the 4-thiouridine residue.

tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.

Affinity Labels↗
PubMed1984

Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, can be crosslinked to 30 S ribosomal protein when the tRNA is placed at the ribosomal A site. This protein has now been identified by immunological methods. The protein-[3H]Phe-tRNA covalent complex, obtained by extraction with 6 M-urea, was reacted separately with each of the 21 purified antisera to 30 S ribosomal proteins. The double antibody technique was used. Anti-S19 was the only antiserum able to precipitate the radioactivity, and 66 to 81% of the added radioactivity could be precipitated. The same result was obtained with three different ribosome preparations, at low as well as high crosslinking yield, with dipeptidyl-tRNA in the A site as well as aminoacyl-tRNA, and when binding and crosslinking were performed at 20 mM-Mg2+ instead of at 5 mM. Therefore, when aminoacyl-tRNA or peptidyl-tRNA is in the ribosomal A site, position 8, which is always uridine or 4-thiouridine, must be within 9 A of protein S19.

Affinity Labels↗
PubMed1983

Formation and properties of a covalent complex between elongation factor Tu and Phe-tRNA bearing a photoaffinity probe on its 3-(3-amino-3-carboxypropyl)uridine residue.

Escherichia coli Phe-tRNA, modified with the photoaffinity reagent 6-(2-nitro-4-azidophenylamino)caproate on the 3-(3-amino-3-carboxypropyl)uridine residue, was crosslinked to E. coli EFTu Section upon irradiation at 0 degree C with visible light at wavelengths greater than 400 nm. Crosslinking was dependent on irradiation, the photoaffinity probe, and was blocked by pre-photolysis. 1 mM-dithiothreitol completely quenched crosslinking. Binding of the tRNA to EFTu was a prerequisite for crosslinking, because neither EFTu . GDP nor AcPhe-tRNA could substitute; EFTu . GDPCP, however, was almost as active as EFTu . GTP. Crosslinking was complete in less than five minutes and was stable to at least 20 minutes of irradiation with a single 650 W tungsten lamp 4 cm away. The crosslinking yield ranged from 15% to 25%. The crosslinked complex possessed several remarkable properties. At 0.5 mM-Mg2+, the complex protected the AA-tRNA link to chemical hydrolysis, stabilized the bound GTP to dissociation or exchange, and was not adsorbed to cellulose nitrate filters. The purified crosslinked complex could be bound to ribosomes with concomitant hydrolysis of GTP. Extensive peptide bond formation with AcPhe-tRNA in the P site occurred despite the presence of the crosslinked EFTu. We conclude that hydrolysis of GTP is sufficient to release the 3' end of the Phe-tRNA from complexation with EFTu. Translocation of the A site bound complex did not occur. The crosslink site on EFTu is probably near the periphery of the molecule, because shortening the probe from 20 A to 14 A completely blocked crosslinking. A similar but shorter 8 A probe, p-azidophenacyl-4-thiouridine located on the opposite face of the tRNA, did not crosslink.

Affinity Labels↗
PubMed1982

E coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl) uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction.

E. coli tRNAPhe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-(4-azido-2-nitrophenyl) glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T1 oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the ribosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on the other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.

Affinity Labels↗
PubMed1982

Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues.

N-Acetylvalyl-tRNA1Val (AcVal-tRNA1Val) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of 300-400 nm. To identify the crosslinked nucleotide in 16S RNA. AcVal-tRNA1Val-16S [32P]RNA was digested completely with RNase T1 and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNA1Val, C1400. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNA1Val-16S RNA prepared from unlabeled ribosomes. This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked T1 oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleotide of tRNA1Val, C-A-C-C-U-C-C-C-U-cmo5U-A-C-m6A-A-G39(cmo5U, 5-carboxymethoxyuridine). A similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16S was cmo5U34, the 5' or wobble base of the anticodon. The adduct is believed to result from formation of a cyclobutane dimer between cmo5U34 of tRNA1Val and C1400 of the 16S RNA.

Base Sequence↗
PubMed1982

Functional conservation near the 3' end of eukaryotic small subunit RNA: photochemical crosslinking of P site-bound acetylvalyl-tRNA to 18S RNA of yeast ribosomes.

Escherichia coli acetylvalyl (AcVal)-tRNA1Val became crosslinked to both yeast and spinach chloroplast ribosomes upon irradiation (300 nm) in the presence of poly(U2,G). Yields were 25-30% and 33%, respectively, compared to 45% for E. coli. Crosslinking occurred to the P site, only to the 40S subunit, and 90% of that was to the 18S rRNA. The crosslink could be photolyzed at 254 nm with the same first-order kinetics as for the E. coli ribosome complex previously studied. The AcVal-tRNA that split off could be crosslinked again when irradiated at 300 nm, showing that the crosslink was photoreversible. There was a strong codon specificity for crosslinking. With pG-U-U, 85% crosslinking was obtained after 20 min of irradiation; with G-U-A, only 3% crosslinking occurred. All of these properties are the same as those previously reported for the E. coli ribosome crosslink that occurs via cyclobutane dimer formation between the 5' anticodon base 5'-carboxymethoxyuridine-34 and cytidine-1400 of the 16S RNA. Cytidine-1400 is in the center of a 17-mer that has been almost totally conserved among the small subunit rRNAs of all species so far examined, including yeast. Crosslinking of tRNA in the same way to both yeast and E. coli ribosomes shows that there has been a functional conservation as well in this region of the small subunit rRNA. This region may be involved in some essential aspect of the decoding process that is common to both prokaryotic and eukaryotic protein synthesis systems.

Base Sequence↗
PubMed1981

Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes.

Escherichia coli 16S RNA from 30S ribosomal subunits was isolated, oxidized at the 3' end, and labeled with the thiosemicarbazide derivatives of fluorescein or eosin. Labeled 16S RNA was reconstituted into 30S subunits. They were almost fully active compared to 30S subunits reconstituted from unlabeled 16S RNA by using a poly(uridylic acid)-directed polyphenylalanine synthesis assay. Fluorophores were placed at three different positions of tRNAPhe. E. coli and yeast tRNAPhe were oxidized at the 3' end and labeled with the thiosemicarbazide derivative of fluorescein or with the hydrazide of N-methylanthranilic acid. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin or 1-aminoanthracene. Also, E. coli tRNAPhe was photochemically cross-linked between 4-thiouridine at position 8 and cytidine at position 13. After reduction, this site was used as a fluorescent probe. The labeled tRNAs were bound into the peptidyl site of 70S ribosomes, and then the distances from the fluorophore in the modified tRNA to the fluorophore at the 3' end of 16S RNA were measured by nonradiative energy transfer. Calculations were based on measurements of fluorescence lifetimes. The distances to the 3' end of 16S RNA were found to be as follows: 3' end of tRNA, 67-74 A; cross-linked t RNA, 53-60 A; anticodon loop of tRNA, greater than 61 A.

Binding Sites↗