The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Beauclerk, A A; Cundliffe, Eric

doi:10.1002/j.1460-2075.1988.tb03236.x

Cited by 42 publications

(32 citation statements)

References 29 publications

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“…Evidence for a participation of L2 in the translational process derives from its attachment site on 23 S rRNA. It has been shown by chemical and ribonuclease footprinting methods that L2 binds to the central part of domain IV on 23 S rRNA [33,41]. This domain, together with domain V of 23 S rRNA, is now generally accepted as being an integral and functional important part of the peptidyltransferase centre [42].…”

Section: Discussionmentioning

confidence: 99%

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Ühlein

Weglöhner

Urlaub

et al. 1998

Biochemical Journal

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The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In i o replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and

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Section: Discussionmentioning

confidence: 99%

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Ühlein

Weglöhner

Urlaub

et al. 1998

Biochemical Journal

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“…According to these studies, the 3Ј-terminal adenosine of Asite tRNA Phe protects G2553, whereas that of P-site tRNA Phe protects U2506, U2584, and U2585. All of these nucleotides are located within or near the central loop of domain V. Several lines of evidence indicate that this region of the 23 S rRNA is located in proximity to proteins L2 and L27, which are positioned on the 50 S subunit interface in the valley between the L1 ridge and the central protuberance (3,38,39). At the same time, the 3Ј-terminal adenosine of the E site tRNA Phe protects G2112, G2116, and C2394.…”

Section: Defining the Nucleotides Of The 23 S Rrna At The Peptidylmentioning

confidence: 99%

Transit of tRNA through the Escherichia coliRibosome

Wower

Kirillov²,

Wower³

et al. 2000

Journal of Biological Chemistry

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When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNAPhe containing 2-azidoadenosine at the 3 terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3 termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA Phe , remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center.In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.

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“…Owing to the paucity of information on the location of tRNA segments other than the 3' end and the anticodon, the ribosomal components that neighbor the central region of the tRNA molecule are rather poorly defined. In our model, we have placed the corners of the L-shaped tRNAs so that their D loops are separated by 35 A, the distance derived from fluorescence energy-transfer measurements (36 highly conserved region whose possible importance in protein synthesis has been until now a matter of speculation (37)(38)(39) (46). Second, tRNA bound to the ribosomal P site was found to protect A1916 and A1918 in the highly methylated loop of domain IV, in addition to several residues in domain V, against chemical modification (47).…”

mentioning

confidence: 99%

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.

Wower

Hixson

Zimmermann

1989

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

111

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Photoreactive derivatives of yeast tRNAh containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identifiled at the peptidyl- The formation and analysis of intermolecular crosslinks provides an effective methodology for delineating the topography of tRNA binding sites on the ribosome (1, 2). The UV-induced attachment of tRNAvW' to the peptidyl (P) site of Escherichia coli ribosomes (3), for instance, led to the demonstration that the 5-carboxymethyoxyuridine at position 34 (cmo5U34), the 5' anticodon base of the tRNA, undergoes cycloaddition with, and is therefore within a few angstroms of, C1", a highly conserved nucleotide near the 3' end of 16S rRNA (4). Further, through the use of immuno electron miroscopy, the site of interaction was shown to lie in the cleft between the head and platform of the 30S ribosomal subunit (5). Precise localization of the decoding site within the ribosome was feasible in large part because of the short length of this crosslink. In contrast, contacts between the 3' end of tRNA and the ribosome at the peptidyltransferase center are still poorly defined. Thus, tRNAs containing chemically or photochemically reactive groups attached to the aminoacyl moiety have been reported to label proteins L2, 11, L14, L15, L16, L18, L23, and L27 of the 50S subunit, proteins S14 and S18 of the 30S subunit, and various segments of the 23S rRNA (2). However, immuno electron microscopic studies indicated that the proteins listed above are scattered over a large fraction of the ribosome surface (6) and clearly cannot all be in the vicinity of the tRNA 3' terminus. The diversity of proteins labeled no doubt derives, at least in part, from the size of the reactive substituents, as the crosslinks they establish are typically 10-20 A in length and may therefore extend to ribosomal components at a considerable distance from the site of tRNA binding.To develop a strategy that would yield short-range crosslinks between tRNA and the peptidyltransferase center approximating that between cmo5U34 in tRNAval and C'" in 16S rRNA, we have selectively incorporated photoreactive azidopurines into yeast tRNAPhC in place of their natural counterparts. In a previous report (7)

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The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Cited by 42 publications

References 29 publications

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Transit of tRNA through the Escherichia coliRibosome

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.

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