Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis

Stebbins-Boaz, Barbara; Gerbi, Susan A.

doi:10.1016/0022-2836(91)90614-c

Cited by 21 publications

(24 citation statements)

References 43 publications

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“…The bases in the helical stem were almost completely inaccessible to single-strand-specific modification in all three species investigated. Exceptions, however, were found at helix termini or next to bulged nucleotides, that is, at positions previously shown to be susceptible to single strand modification, presumably because of increased dynamic instability of the helices at such positions (Stebbins-Boaz and Gerbi 1991). In contrast to the low reactivity to single-strand-specific reagents, the helical stem was accessible to cleavage by RNase V1 in both complementary sequences in all three organisms studied.…”

Section: Secondary Structure Of Es3 and The 3-region Of Es6mentioning

confidence: 72%

Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Alkemar

Nygård²

2004

RNA

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The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

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Section: Secondary Structure Of Es3 and The 3-region Of Es6mentioning

confidence: 72%

Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Alkemar

Nygård²

2004

RNA

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“…Only two residues thought to participate in base pairs were methylated to a significant extent in vivo, and each of these adenosine nucleotides was located at the end of a double helix, where breathing (transient separation) of the terminal base pair might occur. In addition, a few adenosine and cytosine residues likely to be in single-stranded regions of the ompA 5' UTR were resistant to methylation; this phenomenon is commonly observed when RNA is treated with dimethylsulfate (27,46) and may reflect higher-order (e.g., tertiary) structure.…”

Section: Resultsmentioning

confidence: 99%

Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA

et al. 1991

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The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serraia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants.

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“…Possibly, the 2581 region is shielded in the empty ribosome but interacts with the CCA end if a tRNA is bound to the P site. In fact, a structural change (melting) of this region upon association of the small and large subunits has been observed in eukaryotic ribosomes (24).…”

Section: Table Imentioning

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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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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Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis

Cited by 21 publications

References 43 publications

Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA

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

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