UGA suppression by a mutant RNA of the large ribosomal subunit.

Jemiolo, David K.; Pagel, Frances T.; Murgola, Emanuel J.

doi:10.1073/pnas.92.26.12309

Cited by 21 publications

(28 citation statements)

References 49 publications

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“…In addition, the suppression of stop codons can result from the misincorporation of near-cognate tRNAs. The latter mechanism is enhanced by mutations in the ribosome and by drugs that target the ribosome (14,19). In these cases, the modification of the ribosome promotes nonsense suppression by interfering with translation fidelity and/or by decreasing the efficiency of translation termination (27).…”

Section: Discussionmentioning

confidence: 99%

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2

Caban

Kinzy

Copeland

2007

Molecular and Cellular Biology

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The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA [Ser]Sec and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.

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Section: Discussionmentioning

confidence: 99%

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2

Caban

Kinzy

Copeland

2007

Molecular and Cellular Biology

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“…We previously described two mutations that cause readthrough specifically at UGA codons, one at bulge nucleotide 1054 in 16S rRNA (13), the other at loop nucleotide 1093 in 23S rRNA (9). In vitro, both are defective in UGA-dependent hydrolysis of ribosome-bound peptidyl-tRNA (1) and are defective in the binding of release factor 2 (RF2) (2), which functions specifically at the UGA termination codon.…”

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confidence: 99%

Mutations in the GTPase Center of Escherichia coli 23S rRNA Indicate Release Factor 2-Interactive Sites

Pagel

Murgola

2002

J Bacteriol

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Mutations in the GTPase center of Escherichia coli 23S rRNA were characterized in vivo as UGA-specific nonsense suppressors. Some site-directed mutations did not exhibit suppressor activity and were interspersed among suppressor mutations. Our results demonstrate the involvement of the two adjacent loops of this conserved rRNA structure in UGA-dependent translation termination and, taken with previous in vitro analyses and with consideration of the crystal structure of the GTPase center RNA, indicate that nucleotides 1067, 1093, 1094, and 1095 are sites of interaction with release factor 2.

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“…The G1093A mutation was also shown to cause UGA-specific readthrough under certain conditions in vivo (17) and to affect mainly RF2-dependent termination in vitro (1,4). Therefore, these results strongly implicate helix 73 in RF2-dependent termination.…”

Section: Discussionmentioning

confidence: 61%

“…The importance of rRNA for the last step of translation, peptide chain termination, has been demonstrated by in vivo and in vitro experiments (1,4,8,10,17,20,27,34). In particular, the G1093A mutation in domain II of Escherichia coli 23S rRNA (Fig.…”

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confidence: 99%

Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

2002

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Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.The rRNAs from both subunits have been implicated in various stages of translation (3,22,24,25,36). The importance of rRNA for the last step of translation, peptide chain termination, has been demonstrated by in vivo and in vitro experiments (1,4,8,10,17,20,27,34). In particular, the G1093A mutation in domain II of Escherichia coli 23S rRNA (Fig. 1, top), isolated as a codon-specific suppressor of UGA mutations, was shown to cause defects in the catalysis of peptidyltRNA hydrolysis in the presence of release factor 2 (RF2) in a realistic in vitro translation system (1). (RF2 mediates termination at UGA and UAA, while RF1 functions at UAG and UAA [18,29].) In addition to the catalytic defect caused by G1093A, this mutation also affects binding of RF2 to the ribosome (4), clearly suggesting that defective termination contributes to the readthrough of nonsense codons detected in vivo. Nucleotide G1093 is in the highly conserved structure historically referred to as the GTPase center (also the binding region for large-subunit ribosomal protein L11). In this study, we asked if there are sites elsewhere in 23S rRNA that are functionally associated with G1093 during translation termination. These sites could interact either directly or indirectly with G1093. An indirect interaction could, for example, be transmitted by RF2. To identify such sites, we took advantage of the fact that G1093A, when expressed from a multicopy plasmid, is lethal at temperatures above 37°C (21). After DNA segmenttargeted PCR random mut...

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UGA suppression by a mutant RNA of the large ribosomal subunit.

Cited by 21 publications

References 49 publications

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2

Mutations in the GTPase Center of Escherichia coli 23S rRNA Indicate Release Factor 2-Interactive Sites

Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

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