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Chapter 23 : Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center

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Abstract:

The chapter describes an approach to form defined photo-cross-links from targeted RNA sites within the ribosome, with an emphasis on those that are important functionally. In this approach, radioactive, photolabile derivatives of oligonucleotides (PHONTs) having sequences complementary to rRNA sequences are bound to their targeted sequences in intact ribosomal subunits and, on photolysis, form cross-links with neighboring ribosomal components. The PHONT approach offers several advantages. First, it allows targeting of sequences of particular functional or structural significance throughout the ribosome structure. Second, the cross-links formed provide a defined upper-limit distance for the separation of the linked components within the ribosome, given by the length of the tether. The chapter first describes the PHONT approach in general before presenting the results of recent applications of the approach to the study of the peptidyltransferase center (PTC). It identifies four principal elements in PHONT design: first, the backbone structure; second, the placement of the photolabile group within the oligonucleotide sequence; third, the length and flexibility of the tether linking the photolabile group with the oligonucleotide backbone; and fourth, the introduction of radioactivity. As is evident in the descriptions, PHONT design continues to evolve. Currently the YAMMP approach is applied to develop a three-dimensional model of the PTC based on cross-linking results, which provide the clearest set of constraints for model construction.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23

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Figure 1

Space-filling model of heteroduplex between PHONTs and nt 517 to 527 in 16S rRNA. The tethered aryl azides correspond to five different PHONTs targeting this sequence ( ).

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Image of Figure 2
Figure 2

Noncovalent binding of oligonucleotides to 50S subunits. ♦, 2′-OMe-RNA-p*2612-2604; ●, cDNA-p*2612-2604; ▲, 2′- OMe-RNA-p*2258-2253/ 52(S)-2248. The last oligonucleotide is the precursor to PHONT 5 ( Fig. 3 ), in which a nonbridging oxygen on the phosphoryl group connecting nucleotides complementary to G2253 and G2252 is replaced by a sulfur. The error bars indicate average deviations. Asterisks denote P-labeled material.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Image of Figure 3
Figure 3

Structures of PHONTs 1 to 7. The distances between the photogenerated nitrenes (or 4-thio position) and the bases complementary to rRNA nucleotides are indicated. ABA, azidobenzoylamide; SAz, -azidophenacyl derivative of a thiophosphate; *p and A*, P-labeled phosphoryl and adenyl groups placed at the 5′ and 3′ positions, respectively.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Figure 4

RNase H digestion of 50S rRNA labeled by PHONTs 1 and 2. Labeled 50S RNA isolated from 50S subunits photolyzed in the presence of PHONT 1 or 2 was incubated with two equivalents of the indicated cDNA probe(s) and digested with RNase H. The cleavage products were subjected to urea-PAGE and visualized by autoradiography. Photolabeling experiments were carried out either in the absence of competitor 2′-OMe-oligoRNA [lanes (-)] or in the presence of a 10-fold excess (over the 50S subunit) of 2′- OMe-oligoRNA either complementary to nt 2604 to 2612 (PHONT 1 target) or 2448 to 2458 (PHONT 2 target) (lanes CH) or containing mismatches (lanes MM) to these two targets. Lane 1, RNA size markers. Lane 2, DNA size markers, with sizes (in nucleotides) indicated to the left of the gel. RNA sizes are italicized. Lanes 3 to 14, 23S rRNA labeled with photoprobe 1, digested with cDNAs 1892 to 1883 and 2505 to 2497 (lanes 3 to 5); cDNAs 1892 to 1883 and 1971 to 1962 (lanes 6 to 8); cDNAs 866 to 857 and 1051 to 1042 (lanes 9 to 11); cDNAs 866 to 857 and 916 to 907 (lanes 12 to 14). Lanes 15 to 17, 23S rRNA labeled with photoprobe 2, digested with cDNAs 2505 to 2497 and 2310 to 2301. The arrows point to specifically labeled RNase H fragments.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Figure 5

Reverse transcriptase analyses of PHONT 1-labeled 23S RNA extracted from PHONT 1-labeled 50S subunits. Lanes U, C, G, and A, sequencing products generated from control (nonphotolyzed) 23S rRNA in the presence of ddATP, ddGTP, ddCTP, and ddTTP, respectively. Lanes 1 to 3, control experiments for rRNA isolated from samples with (+) or without (−) photolysis (hν) as indicated. Lanes 4 to 6, samples photolyzed with PHONT 1 in the presence or absence of complementary (CH) or mismatched (MM) 2′-OMe-oligoRNAs as indicated. The arrows point to nucleotides at which pauses or stops induced by photoincorporation of PHONT 1 are observed. (A) With primer complementary to 23S rRNA nt 2639 to 2623. (B) With primer complementary to 23S rRNA nt 1983 to 1965.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Figure 6

Analyses of PHONT 1-labeled TP50. (A) RP-HPLC. TP50 was extracted from 50S subunits labeled with PHONT 1 (2′-OMe-RNA-p*2612-2604- C7-Az) in the absence or presence of either competitive complementary 2′-OMe–oligoRNA (2′-OMe-RNA-2612-2604) or MM-2′-OMe-RNA-2612-2604). (B) SDS-PAGE and autoradiographic analyses of RP-HPLC peak C in panel A. The lefthand four lanes represent TP50; the right-hand three lanes represent peak C. Lane (-), 50S subunits were incubated with prephotolyzed 1 with no further photolysis. Lanes (+), subunits photolyzed with 1. Lanes CH and MM, subunits photolyzed with 1 in the presence of 10-fold excess (over 50S subunits) of either 2′-OMe-RNA-2612-2604 (lanes CH) or MM-2′-OMe-RNA- 2612-2604 (lanes MM). The far righthand lane displays TP50 stained with Coomassie blue.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Figure 7

Oligonucleotides testing the target site specificity of PHONT 5 photoincorporation. The asterisk indicates the position of aryl azide attachment. Underlined nucleotides are not complementary to the target site.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Figure 8

Summary of cross-linking results useful for construction of a peptidyltransferase model. (A) The circled cross-links are from Table 1 . The large boldface numbers refer to target sites for PHONTs 1 to 6. (B) All relevant cross-links. The circled cross-links are as described in panel A. The boxed cross-links involve a variety of other approaches, mostly direct photolysis or via introduction of a photolabile group. The helices (underlined) are numbered as in Brimacombe, 1995.

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23
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Tables

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Table 1

Site-specific cross-links from PHONTs for 23S rRNA

Citation: Cooperman B, Vladimirov S, Druzina Z, Seo H, Bukhtiyarov Y, Wang R. 2000. Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center, p 271-286. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch23

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