Das 3′‐Ende der tRNA und seine Rolle bei der Proteinbiosynthese

Chládek, Stanislav; Sprinzl, Mathias

doi:10.1002/ange.19850970506

Cited by 18 publications

(6 citation statements)

References 185 publications

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“…The specific stimulation seen with CMP (Fig. 5) was interpreted that the universal C75 is essential for the proper binding of the donor substrate at the PTF center (11,14). The intriguing finding is that the residual activities observed with ribosomes with the mutants G2581A and ⌿2580C cannot be stimulated with CMP (Fig.…”

Section: Table Imentioning

confidence: 97%

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Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

Spahn

Schäfer

Krayevsky

et al. 1996

Journal of Biological Chemistry

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Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence ⌿GG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schä fer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849 -32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and ⌿2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.Two out of the 55 UGG sequences in 23 S rRNA from Escherichia coli are universally conserved in the non-mitochondrial rRNAs of the large ribosomal subunit, UGG2251 and ⌿GG2582. The first two positions of both sequences were mutated, and the effects on ribosomal assembly and functions were analyzed (1). Mutants of the first sequence did not influence ribosomal assembly and bacterial growth but caused some defects in in vitro poly(U) translation. Those of the second sequence are characterized by a seemingly normal assembly but with severe defects in peptide synthesis (1). The ⌿GG2582 sequence is located at the "peptidyltransferase ring" (see Fig. 1). Here we analyze the peptidyltransferase (PTF) 1 activity in various systems and the translocation reaction. All mutant ribosomes were able to translocate. The helix 80 mutants show a normal puromycin reaction. In contrast, the helix 90 mutations severely interfere with the PTF activity. MATERIALS AND METHODSPlasmids and strains, the preparation of ribosomes and polysomes, and the quantification of plasmid-born 23 S rRNA have been described in the preceding paper (1).Puromycin Kinetics with AcPhe-tRNA Bound to the P-sites of Poly(U)-programmed 70 S-tRNA binding and puromycin reaction was essentially as described (2) except that the buffer conditions (20 mM Hepes-KOH (pH 7.6), 6 mM MgCl 2 , 150 mM NH 4 Cl, 4 mM ␤-mercaptoethanol, 2 mM spermidine, and 0.05 mM spermine) were kept constant in all steps (3). 70 S ribosomes were programmed with Ac[14 C]Phe-tRNA Phe (1,030 dpm/pmol, 1.5-fold excess over 70 S) and poly(U). After an incubation at 37°C for 30 min, two aliquots were filtered over nitrocellulose to determine the binding. To the remaining aliquots, puromycin was added, and the kinetics of AcPhe-puromycin formation were follow...

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Section: Table Imentioning

confidence: 97%

“…The interpretation was that C75 of the universally conserved CCA-3Ј end of the tRNAs plays a critical role for the binding of the peptidyl-residue at the PTF center (11,14).…”

Section: Fig 2 Kinetics Of the Puromycin Reaction Acphe-trnamentioning

confidence: 99%

Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

Spahn

Schäfer

Krayevsky

et al. 1996

Journal of Biological Chemistry

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“…Freie benachbarte OH-Gruppen am 3'-Ende der Aminoacyl-und PeptidyltRNA sind mögliche Protonendonoren; [5] dies gab Anlass, die 2',3'-Positionsisomere der Aminoacyl-und Peptidyl-tRNAs unter Berücksichtigung der Bindung des Acylrestes am 2'-oder 3'-OH-Rest sowie die Anforderungen an die freien benachbarten OH-Gruppen während der Translation umfassend zu untersuchen. [6][7][8] Für einen erfolgreichen Peptidtransfer durch die Peptidyltransferase müssen sowohl der Aminoacylrest in der AStelle als auch der Peptidylrest in der P-Stelle an die 3'-OHGruppe des terminalen Adenosinrestes der jeweiligen tRNA gebunden werden. Das Fehlen der benachbarten 2'-OHGruppe der Aminoacyl-tRNA beeinflusst den Peptidtransfer nicht; die Rolle der 2'-OH-Gruppe der Peptidyl-tRNA ist hingegen unklar.…”

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Synthese der Peptidbindung am Ribosom: keine freie benachbarte Hydroxygruppe an der terminalen Ribose der Peptidyl‐tRNA erforderlich

2008

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“…Base pairing of the tRNA CCA-3Ј-end with a complementary UGG sequence of 23 S rRNA is the most likely explanation for experimental data gained with minimal substrates (25,26) and with tRNAs mutated in the CCA-3Ј-end (27) as substrates of the P-site region of the PTF center. The 23 S rRNA contains 55 UGG sequences, only two of which are universally conserved in non-mitochondrial 23 S-type rRNA, and are therefore candidates for binding the universal CCA-3Ј-end of tRNAs, possibly via canonical base pairing.…”

mentioning

confidence: 99%

Mutational Analysis of Two Highly Conserved UGG Sequences of 23 S rRNA from Escherichia coli

Spahn

Rèmme

Schäfer

et al. 1996

Journal of Biological Chemistry

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The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (⌿2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.The central enzymatic activity of ribosomes is the formation of peptide bonds. The corresponding peptidyltransferase (PTF) 1 center is located on the large ribosomal subunit (1, 2). Reconstitution analyses have identified the ribosomal proteins L2, L3, and L4, and the 23 S rRNA as PTF candidates in Escherichia coli ribosomes (3-5). A complex derived from the large subunit of Thermus aquaticus ribosomes consisting of 23 S rRNA and only 3-8 proteins had significant PTF activity (6, 7). This observation underscores the possible involvement of 23 S rRNA in this activity.An impressive wealth of data points to a distinct feature of the secondary structure map of 23 S rRNA, the so-called "peptidyltransferase ring" of domain V (8, 9) as a component at or near the PTF center. The PTF ring comprises about 40 nucleotides and represents a cluster of universally conserved nucleotides (10, 11). It is satisfying that a highly divergent array of methods correspondingly identifies the same region of 23 S rRNA, methods such as cross-linking studies with substrates of the PTF center (12-15), mutations of the 23 S rRNA gene conferring resistance against inhibitors of the PTF activity (for review, see Refs. 16 and 17), and protection studies (18 -20).Some studies aimed to identify nucleotides at the PTF center. The nucleotides G2252 and G2253, which are at the helixloop 80, were protected against kethoxal by the 3Ј-terminal CCA of P-site bound tRNA (19). Mutation of G2252 generated a dominant lethal phenotype (21). A double mutant having both Gs altered was lethal and showed a reduced activity of peptide bond formation by approximately 50% (22). A recent analysis presented evidence that G2252 is involved in canonical base pairing with C74 at the acceptor end of tRNA, thus obviously playing a role in the binding of the donor substrate at the P site region of the PTF center (23).Another study applied a random mutagenesis of the "Southern half" of the PTF ring (residues 2493-2606). With an elegant screening procedure, 21 mutations of 18 positions were found. Mutatio...

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Das 3′‐Ende der tRNA und seine Rolle bei der Proteinbiosynthese

Cited by 18 publications

References 185 publications

Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

Conserved Nucleotides of 23 S rRNA Located at the Ribosomal Peptidyltransferase Center

Synthese der Peptidbindung am Ribosom: keine freie benachbarte Hydroxygruppe an der terminalen Ribose der Peptidyl‐tRNA erforderlich

Mutational Analysis of Two Highly Conserved UGG Sequences of 23 S rRNA from Escherichia coli

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