Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

Velichutina, Irina; Dresios, John; Hong, Joo Y.; Li, Chibo; Mankin, Alexander S.; Synetos, Dennis; Liebman, Susan W.

doi:10.1017/s1355838200000637

Cited by 49 publications

(51 citation statements)

References 46 publications

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“…This bias seems to be due to the use of XL1-Red, because transversions engineered at several of these positions confer strong suppressor phenotypes that would have been easily detected in the screens (see below). Of the 34 mutations recovered, at least six (G886A, U911C, C1054U, C1200U, C1469U, and G1491A) were shown previously to influence the fidelity of elongation (Allen and Noller 1991;Gregory and Dahlberg 1995;Murgola et al 1995;Lodmell and Dahlberg 1997;Pagel et al 1997;Velichutina et al 2000).…”

Section: Resultsmentioning

confidence: 93%

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

McClory¹,

Leisring²,

Qin³

et al. 2010

RNA

126

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The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.

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Section: Resultsmentioning

confidence: 93%

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

McClory¹,

Leisring²,

Qin³

et al. 2010

RNA

126

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“…These occurred at nucleotides G1168-G1170 (G886-888 in E. coli), which reside near to the decoding center of the small subunit (Wimberly et al 2000). Mutations in yeast ribosomes at nucleotides 886 and 888 (E. coli numbering) lead to phenotypes of increased accuracy (Velichutina et al 2000). The fact that binding of the HCV IRES RNA results in changes in the chemical reactivity of these nucleotides suggests that the IRES could have an effect on the decoding center of the ribosome.…”

Section: Wwwrnajournalorg 1901mentioning

confidence: 99%

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Shenvi¹,

Dong²,

Friedman³

et al. 2005

RNA

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Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg 2+ is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg 2+ concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg 2+ that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg 2+ concentrations. In low Mg 2+ concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.

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“…As a result, [PSI + ] strains experience an increased level of readthrough due to the limitation of functional eRF3. In addition, mutations within the small subunit (18S) and large subunit (25S) ribosomal RNAs have also been shown to cause an increased rate of translational readthrough in yeast (Chernoff et al 1994;Liu and Liebman 1996;Velichutina et al 2000Velichutina et al , 2001.…”

Section: Introductionmentioning

confidence: 99%

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae

Keeling¹,

Lanier²,

Du³

et al. 2004

RNA

159

170

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The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1⌬ strain and a [PSI + ] strain using a collection of translation termination reporter constructs. The [PSI + ] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3 of the stop codon. We found that the upf1⌬ mutation, like the [PSI + ] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI + ]/upf1⌬ strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1⌬ mutation and the [PSI + ] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was ≥ 0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was ≤ 0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.

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Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

Cited by 49 publications

References 46 publications

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae

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