Neighbourhood of the central fold of the tRNA molecule bound to the<i>E.coli</i>ribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Podkowiński, Jan; Górnicki, Piotr

doi:10.1093/nar/19.4.801

Cited by 20 publications

(10 citation statements)

References 26 publications

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“…6). A further connection is protein L33, which has been crosslinked to helix 88 (29) as well as to the 3' terminus of E-site-bound tRNA (36), and to positions 20a (37) and 47 (31) of P-site-bound tRNA, the latter position in turn having a crosslink site (31) in helix 84. The majority of the footprint sites for P-or A-site-bound tRNA (38) also lie within this general area of the 23S RNA.…”

Section: Resultsmentioning

confidence: 99%

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Dontsova

Тишков

Dokudovskaya

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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A DNA fragment containg the Escherichia coUl 5S rDNA sequence linked to a 17 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with ¶7 polymerase using a mixture of [a-32P]UTP and 4-thio-UTP, yielding a transcript in which 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.Interactions and neighborhoods between 5S rRNA and other components of the Escherichia coli 50S ribosomal subunit have been investigated in a number of laboratories by a variety of techniques. From protein binding studies (e.g., refs. 1 and 2) the 5S RNA has long been known to interact with proteins L5, L18, and L25, and the binding sites of these proteins on the 5S molecule have been studied in detail by footprinting methods (e.g., refs. 3 and 4). The 5' and 3' ends of the 5S RNA (5-7), as well as residues A-39 and U-40 at the loop end of helix III (8), have been localized by immunoelectron microscopy (IEM) on the central protuberance ofthe 50S subunit, in good agreement with the corresponding IEM locations ofproteins L5, L18, and L25, all of which were also found to lie on the central protuberance (9, 10). On the other hand, very little information is available relating to possible interactions between 5S and 23S RNA (11).Our laboratories have made extensive use of mRNA analogues containing 4-thiouridine (thio-U) residues, in order to study contacts between mRNA and 16S RNA by photocrosslinking (12-15), and the success of this approach has prompted us to apply the same methodology in the search for contacts between 5S and 23S RNA. Accordingly, a modified 5S RNA was constructed containing a random distribution of thio-U residues in place of normal uridine, and the modified molecule was reconstituted into 50S subunits or 70S ribosomes as a substrate for photocrosslinking. The principal result of the subsequent analysis, which we report here, was the characterization of a high-yield crosslink from the loop end of helix IV of the 5S RNA to a site close to the peptidyltransferase ring of the 23S RNA.MATERIALS AND METHODS Construction of Modified 5S RNA. Plasmid pKK223-3 (Pharmacia), which carries the E. coli 5S rDNA sequence, was cut with HindIII and Ssp I to yield a 500-bp fragment encompassing the 5S rDNA; this fragment was cloned by standard procedures (16) into phage M13mpl8 (...

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Section: Resultsmentioning

confidence: 99%

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Dontsova

Тишков

Dokudovskaya

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Helix 80 is adjacent to helix 75 where the N domain of L9 binds ( Figure 6). Close proximity of the dihydrouridine loop (tRNA elbow) of P-site tRNA to L1 has been demonstrated with long-range crosslinking reagents of 10 and 19 Å at nt 20 (Podkowinski & Gornicki, 1991) and a shorter range reagent (4-thiouridine, 2 to 4 Å ) at nt 17 of the tRNA (Rosen et al, 1993). In addition, nt 8 of P-site tRNA (on the inside of the elbow) has been crosslinked to nt 2111 of the 23 S rRNA, using 4-thiouridine (Rinke-Appel et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Adamski

Atkins

Gesteland

1996

Journal of Molecular Biology

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“…sites of mutations causing resistance to antibiotics (e.g. Sigmund et al, 1984;Melancon et al, 1988;Powers and Noller, 1991), sites of mutations affecting the termination of translation (Murgola et al, 1988;Shen and Fox, 1989), and the sites of footprinting to P-or A-site bound tRNA (Moazed and Noller, 1990;Figure 6). (In the latter instance it is worth noting that the footprint observed for A-site bound tRNA may in fact have been due to the mRNA rather than the tRNA.)…”

Section: Discussionmentioning

confidence: 99%

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

et al. 1992

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Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio‐uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio‐U residues were activated by UV irradiation and the resulting sites of cross‐linking to 16S RNA analysed. Three distinct cross‐links were consistently observed: (i) from position ‘+6’ of the mRNA (the 3′‐base of the A‐site codon) to base 1052 of 16S RNA; (ii) from position ‘+7’ of the mRNA to base 1395; and (iii) from ‘+11’ to base 532. Individual yields of the cross‐links were strongly dependent on the particular mRNA sequence in each case. The ‘+11/532’ and ‘+6/1052’ cross‐links were always entirely tRNA‐dependent, whereas the ‘+7/1395’ cross‐link was observed at lower intensity in the absence of tRNA. In the presence of a second (A‐site bound) tRNA the +6/1052 cross‐link was markedly reduced. A cross‐link to the 1050 region was again observed when a message carrying a thio‐U at position ‘+9’ was translocated on the ribosome so as to bring the thio‐U to position +6. Taken together, the data are incompatible with some current models both for the three‐dimensional arrangement of 16S RNA and for the orientation of the tRNA‐mRNA complex in the ribosome.

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Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

Cited by 20 publications

References 26 publications

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

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