[54] Photo-induced cross-linking of 4Srd and Cyd residues in Escherichia coli tRNA and its use as a conformational probe

Ofengand, James; Delaney, Pamela; Bierbauivi, J.

doi:10.1016/0076-6879(74)29059-1

Cited by 7 publications

(6 citation statements)

References 17 publications

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“…In order to alkylate specifically the s2C residue at position 32 of tRNAfrg, it was first necessary to sequester s4U(8), which also reacts with the photoaffinity reagents used in this work. To this end, s4U(8) was internally cross-linked to C( 13) by irradiation of the tRNA at 350 nm (Ofengand et al, 1974). Reduction with NaBH4 converts the photoproduct to a strongly fluorescent derivative, providing a convenient assay for the rate and extent of internal cross-link formation (see Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, we concluded that both thio bases could be alkylated to about the same extent by IA and APAA-I (see Figure 1 and Table II). Derivatization could be directed specifically to s2C(32), however, by initially establishing an internal cross-link between s4U(8) and C(13) via irradiation at 350 nm (Ofengand et al, 1974). A second difference concerns the aminoacylation of tRNAArg after modification of s2C(32).…”

Section: Discussionmentioning

confidence: 99%

“…Internal Cross-Linking of tRNAs. Internal cross-links between residues s4U(8) and C(13) in tRNAfrg and tRNA/al were formed by irradiation at 350 nm and subsequent reduction with sodium borohydride (Ofengand et al, 1974). The fluorescence of the reduced, cross-linked bases was monitored with a Farrand spectrofluorometer using excitation and 1 Abbreviations: cmo5U, 5-(carboxymethoxy)uridine; s2C, 2-thiocytidine; s4U, 4-thiouridine; AcArg, TV-acetylarginyl; Hepes, 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; Bicine, A,Ar-bis(2-hydroxyethyl)glycine; APA-Br, p-azidophenacyl bromide; APAA-I, p-azidophenacyl iodoacetate; NAMA-I, iV-(4-azido-2-nitrophenyl)-A'-methyl-2-aminoethyl iodoacetate; IA, iodoacetamide; Me2SO, dimethyl sulfoxide; (XL)tRNA, tRNA with an internal cross-link between s4U(8) and C( 13 [3H]APAA-I (74 mCi/mmol) were prepared as previously described (Ofengand et al, 1977).…”

Section: Methodsmentioning

confidence: 99%

“…One was reduced with sodium borohydride and assayed for fluorescence at 440 nm (Ofengand et al, 1974). The other was tested for the incorporation of [14C]iodoacetamide as described under Experimental…”

Section: T H Imentioning

confidence: 99%

“…Previous modification of s2C(32) with monofunctional alkyl halides indicated that the presence of the substituents produced only minor alterations in the biological activity of the tRNA (Kruse et al, 1978). In the present study, we have attached three bifunctional photoaffinity probes of different lengths to s2C(32) in tRNAfrg after first inactivating the s4U residue at position 8 by internally cross-linking it to C(13) (Ofengand et al, 1974). AcArg-tRNAfrg derivatives containing these modifications were then cross-linked to the P site of 70S ribosomes by irradiation, and the ribosomal components thus labeled were characterized.…”

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confidence: 96%

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Photochemical crosslinking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop

Chen¹,

Franke²,

Hixson³

et al. 1985

Biochemistry

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The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: T H Imentioning

confidence: 99%

mentioning

confidence: 96%

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Photochemical crosslinking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop

Chen¹,

Franke²,

Hixson³

et al. 1985

Biochemistry

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The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

et al. 1994

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A photo‐reactive diazirine derivative was attached to the 2‐thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo‐activation of the diazirine label, the sites of cross‐linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross‐linking. From tRNA at the A site, a major cross‐link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross‐links to positions 693 and to 957 and/or 966, as well as a minor cross‐link to position 1338. The E site bound tRNA showed major cross‐links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross‐link observed from the A site). Immunological analysis of the concomitantly cross‐linked ribosomal proteins indicated that S7 was the major target of cross‐linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.

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A Model of the tRNA Binding Sites on the Escherichia Coli Ribosome

Wower

Sylvers

Rosen

et al. 1993

The Translational Apparatus

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[54] Photo-induced cross-linking of 4Srd and Cyd residues in Escherichia coli tRNA and its use as a conformational probe

Cited by 7 publications

References 17 publications

Photochemical crosslinking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop

Photochemical crosslinking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A Model of the tRNA Binding Sites on the Escherichia Coli Ribosome

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