Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in<i>Escherichia coli</i>

Abdi, Nimo M.; Fredrick, Kurt

doi:10.1261/rna.2118105

Cited by 61 publications

(90 citation statements)

References 48 publications

(46 reference statements)

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“…Isolation of missense and nonsense suppressor mutations in 16S rRNA Using a specialized ribosome system described previously (Abdi and Fredrick 2005;Qin et al 2007;Qin and Fredrick 2009), a classical genetic approach was taken to identify mutations in 16S rRNA that decrease the fidelity of translation elongation. Two screens were performed, one for missense suppressors and the other for nonsense (UGA) suppressors.…”

Section: Resultsmentioning

confidence: 99%

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

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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: 99%

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

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126

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“…Individual 16S rRNA mutations were introduced by fragment exchange by using restriction enzymes KpnI and CpoI into the pKF207 plasmid, which contains, under the control of an arabinose-inducible promoter, the 16S rRNA gene with a mutated anti-Shine-Dalgarno sequence 5Ј-GGGGU-3Ј (17,18). KLF10 cells [F Ϫ ara ⌬(gpt Ϫ lac)5 (⌽ P ant -SD AUCCC -lacZ) Kan R srlR301::Tn10 ⌬(recA-srl)306] that carry a chromosomally encoded lacZ reporter gene with an altered Shine-Dalgarno sequence (5Ј-AUCCC-3Ј) were transformed with the resulting plasmids and were plated onto LB͞agar plates supplemented with 100 g of ampicillin.…”

Section: Methodsmentioning

confidence: 99%

“…These mutations were engineered in plasmid pKF207, which codes for the 16S rRNA gene with an altered anti-Shine-Dalgarno sequence (GGGGU), and mutant 16S rRNAs were expressed in E. coli strain KLF10, which carries a chromosomal copy of the ␤-galactosidase gene (lacZ) with a ribosome binding site (AUCCC) recognized by pKF207-encoded 16S rRNA (18). The 30S subunits assembled with the plasmid-encoded 16S rRNA translate only lacZ but not other cellular genes.…”

Section: Effect Of Selected Rrna Mutations On Protein Synthesismentioning

confidence: 99%

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Yassin

Fredrick

Mankin

2005

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

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Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.16S rRNA ͉ 30S subunit ͉ resistance ͉ ribosome ͉ translation W ith a molecular mass of Ϸ2.5 million Da and Ͼ50 different RNA and protein building blocks, the ribosome represents one of the largest and most complex enzymes in the cell. Ribosomal RNA (rRNA) accounts for two-thirds of the ribosome and is responsible for its main functions in protein synthesis: interpretation of genetic information and polymerization of amino acids into a polypeptide. rRNA is also intimately involved in known auxiliary activities of the ribosome, such as nascent peptide release, binding of translation factors, GTP hydrolysis, etc. (reviewed in ref. 1).The ribosome is the predominant antibiotic target in the bacterial cell. A large variety of natural and synthetic antibiotics interfere with translation by binding to rRNA and preventing the correct placement of ribosomal ligands, corrupting rRNA structure, or affecting conformational flexibility of rRNA (2). Advantages of the ribosome as an antibiotic target may stem from its RNA-based design. The multiplicity of rRNA genes in microbial genomes makes it difficult for a microorganism to develop resistance by mutating the drug-binding site (3). Furthermore, RNA offers fewer mutational options than protein enzymes (3 versus 19, respectively), which makes it more difficult for a microbial pathogen to ''find'' a mutation that would reduce antibiotic binding without compromising functional integrity of the enzyme. In the clinical setting, ...

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“…Our findings indicate that mutation of C1400 to an A or G resulted in 17% and 16% function, respectively, and that ribosomes containing the C1400U mutation, produced 30% more GFP than the wildtype ribosomes. Abdi and Fredrick found ribosomes expressing C1400 to A or G produced approximately 5% and 9% as much β-galactosidase as wild-type ribosomes, respectively, and that C1400U-containing ribosomes were approximately 50% as active as wild-type ribosomes 23 Hui et al, using a specialized ribosome system with a different SD-ASD combination and with human growth hormone (hGH) as the reporter, found that A1400 and G1400 mutations produced ribosomes that were 50% and 20% as active as wild-type ribosomes and that the U1400 mutation had no effect (100%) on the amount of hGH produced. 48 In vitro analyses of reconstituted ribosomes containing single mutations at position 1400 revealed little effect on subunit association and P-site-tRNA binding, but the mutants showed a significant increase in poly(Phe-Val) synthesis.…”

Section: Importance Of the 966 967 And 968 Stackmentioning

confidence: 99%

“…22 Abdi and Fredrick, however, found that any mutation at position 966 resulted in a loss of ribosomal function (see Discussion). 23 Crystal structures of Thermus thermophilus 30S subunits 24 show interactions between the 970 loop and ribosomal proteins S9, S10, and S13. Residues m 2 G966, m 5 C967 and A968 make backbone contacts with protein S9 24 , A964 and A969 make backbone contacts with protein S10 24 , and U965, A969, and C970 make base contacts with protein S13 24 .…”

Section: Introductionmentioning

confidence: 99%

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

Saraiya¹,

Lamichhane

Chow

et al. 2008

Journal of Molecular Biology

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SummaryThe 970 loop (helix 31) of Escherichia coli 16S rRNA contains two modified nucleotides, m 2 G966 and m 5 C967. Positions A964, A969 and C970 are conserved among the Bacteria, Archaea and Eukarya. The nucleotides present at positions 965, 966, 967, 968 and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this rRNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Non-random nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m 2 G966 or m 5 C967 produce more protein in vivo than wild-type ribosomes. Over-expression of initiation factor 3 (IF3) specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for IF3 binding and for the proper initiation of protein synthesis.

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Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation inEscherichia coli

Cited by 61 publications

References 48 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

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

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