Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre

Barta, Andrea; Kuechler, Ernst

doi:10.1016/0014-5793(83)80844-8

Cited by 16 publications

(13 citation statements)

References 18 publications

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“…The sites of photoreaction can therefore be assigned to positions uridine-2584 and uridine-2585. The involvement of uridine residues is in agreement with our previous identification of the modified nucleotide from alkaline hydrolysis of photoaffinity-labeled 23S RNA (9). The photoaffinity-labeled uridine residues are located in the central loop of domain V of 23S rRNA (Fig.…”

Section: Discussionsupporting

confidence: 77%

“…1) as a probe of the peptidyl transferase region (7)(8)(9). The electron pair of the carbonyl group of the benzophenone moiety can be activated by UV irradiation at 320 nm to react with the C,-H bond of peptides as well as with nucleic acids (9,10).…”

mentioning

confidence: 99%

“…Limited hydrolysis of the photoaffinity-labeled 23S RNA with RNase A demonstrates that the crosslink occurs at a site in the 11S RNA fragment, which comprises the 3'-proximal 1100 nucleotides of 23S RNA. After photoreaction, this complex retains its ability to participate in peptide-bond formation (9). The labeled region of the 23S RNA must therefore reside within the peptidyl transferase center.…”

mentioning

confidence: 99%

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Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

Barta¹,

Steiner²,

Brosius³

et al. 1984

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

134

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ABSTRACT3-(4'-Benzoylphenyl)propionyl[3H]Ph etRNA bound to the peptidyl site of the ribosome is photocrosslinked exclusively to 23S RNA on irradiation at 320 nm. The site of reaction has been identified both by hybridization and primer-extension experiments as uridine-2584 and uridine-2585, located within the central loop of domain V according to the secondary structure model of 23S RNA. The fact that the covalently crosslinked tRNA retains its ability to form a peptide bond, together with the proximity of this site to the position of several mutations leading to chloramphenicol or erythromycin resistance strongly argue that this region of the 23S-like rRNAs is an integral component of the peptidyl transferase site. On the basis of these results, and from comparative analysis of the 16 available large subunit rRNA sequences, we propose a model for the functional organization of the peptidyl transferase site involving interaction of domains II and V of 23S rRNA.The three-dimensional structure of the ribosome still presents one of the most challenging problems in molecular biology. On the ribosome, a variety of events must take place in a coordinate fashion to translate the information provided by the mRNA into the correct amino acid sequence. To define the mechanism of translation, attention so far has mainly focused on the protein components of the ribosome, although from our current knowledge, it is difficult to describe the dynamics of the ribosomal machinery in terms of protein functions alone. There is, however, accumulating evidence that structural interactions between the RNA componentsi.e., rRNAs, tRNAs, and mRNA-play an important role in ribosomal function (1, 2). It is generally assumed that the ribosome provides a favorable environment for peptide-bond formation, the prerequisite for this reaction being the correct alignment of the two tRNAs and the close proximity of their CCA-terminal moieties. Therefore, the elucidation of the architecture of the peptidyl transferase center is of utmost importance for the understanding of the mechanism of protein synthesis.A variety of chemically or photochemically reactive derivatives of aminoacyl tRNA and antibiotics have been used to investigate the ribosomal peptidyl transferase site (for reviews see refs. 3-6). Depending on the derivative used, several ribosomal proteins and/or 23S RNA are affinity-labeled. However, it has not been possible until now to assign a specific peptidyl transferase function to one of the ribosomal proteins. Furthermore, no site of crosslinking of such probes to 23S RNA has been unambiguously positioned in the rRNA sequence.In previous experiments, we used the 3-(4'-benzoylphenyl)propionyl derivative of Phe-tRNA (BP-Phe-tRNA; Fig. 1 electron pair of the carbonyl group of the benzophenone moiety can be activated by UV irradiation at 320 nm to react with the C,-H bond of peptides as well as with nucleic acids (9, 10). BP-[3H]Phe-tRNA bound to the peptidyl site (P site) of poly(U)-programmed ribosomes crosslinks in high yield to the 23S R...

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Section: Discussionsupporting

confidence: 77%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

Barta¹,

Steiner²,

Brosius³

et al. 1984

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

134

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“…In previous experiments, we developed a photo-affinity label for identifying components at or close to the peptidyl transferase region (Barta et al, 1975;Barta and Kuechler, 1983;Barta et al, 1984). Use was made of a benzophenone derivative [3-(4'-benzoylphenyl)propionic acid] covalently attached to the amino group of the Phe moiety of Phe-tRNA.…”

Section: Introductionmentioning

confidence: 99%

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

1988

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Photo‐reactive 3‐(4′‐benzoylphenyl)propionyl‐Phe‐tRNA bound to the A‐ or the P‐site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U‐2584 and U‐2585 at the A‐site and A‐2451 and C‐2452 at the P‐site. Minor crosslinks from both sites were observed at nucleotides A‐2503 to U‐2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo‐crosslinking. In addition, tetracycline was also shown to photo‐crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A‐ and P‐site.

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“…Already well documented is the role rRNA plays in mRNA initiation (2) and tRNA binding (3)(4)(5)(6). In addition, some rRNA sites have been implicated in the peptidyltransferase center (7), and subunit-subunit interactions (8)(9)(10).…”

mentioning

confidence: 99%

Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.

Tapprich¹,

Hill²

1986

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

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The functional involvement of rRNA in the protein translation cycle is becoming increasingly apparent (1). Already well documented is the role rRNA plays in mRNA initiation (2) and tRNA binding (3)(4)(5)(6). In addition, some rRNA sites have been implicated in the peptidyltransferase center (7), and subunit-subunit interactions (8-10).Exposed regions of the rRNA have been probed using chemical modification and nuclease digestion techniques. Examples include guanine-specific kethoxal (11), adeninespecific dimethyl sulfate (12), and base-specific ribonuclease T1 (10). A kethoxal-reactive site, guanosine-791, in the 30S subunit was found to be protected from modification upon 70S ribosome formation (13), suggesting that this site might be utilized in subunit association. A subsequent kethoxal modification study showed that when this base was modified, the ability of the subunits to associate was impaired (8).Additional evidence that the stem and loop structure surrounding guanosine-791 is accessible was shown by mapping the rRNA binding sites of the 16S central-domainbinding proteins (14). These results, coupled with those from chemical modification studies suggest that the single-strand loop 787-795 is exposed, most likely in the interface region where the 50S subunit binds to the 30S subunit, and this rRNA region is apparently functional during ribosomal subunit association.We have recently begun probing various rRNA regions using chemically synthesized DNA oligomers that are complementary to specific regions of the rRNA. Successful hybridization of these cDNA probes to rRNA in situ demonstrates the accessibility of the RNA region on the surface of the ribosome. The function of the exposed region can also be assayed by observing disruption of discrete ribosomal MATERIALS AND METHODSRibosomes were prepared from Escherichia coli strain MRE 600 by the methods of Hill et al. (17). Ribosomal subunits were then purified using zonal centrifugation as outlined by Tam and Hill (18). The purified subunits were stored in small aliquots at -70'C.The DNA probe [d(GTATCTAAT)] was chemically synthesized using phosphotriester chemistry on a Bachem DNA synthesizer. Synthetic DNA from which the blocking groups had been removed was purified on a 2.0 x 40 cm Sephadex G-25 column followed by gel electrophoresis and elution of the full length oligomer from the gel (19). Oligomers were stored until use at -70°C. Purified DNA probe was 5'-end labeled using [y-32P]

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Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre

Cited by 16 publications

References 18 publications

Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.

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