A study of the enzymic transfer of aminoacyl-RNA to Escherichia coli ribosomes

Ravel, Joanne M.; Shorey, RoseAnn L.; Froehner, Stanley C.; Shive, William

doi:10.1016/0003-9861(68)90609-7

Cited by 89 publications

(31 citation statements)

References 20 publications

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“…The extract of E. coli strain A19, harvested at the mid-log phase (0.6A 600 ), was prepared as described (23) and applied to a DEAE-Sepharose column at 50 mM NH4Cl. Fractions eluted with 400 mM NH 4 Cl were pooled and used as E. coli aaRS after dialysis against the initial buffer (23).…”

Section: Materals and Methodsmentioning

confidence: 99%

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA Met with the anticodon f 5 CAU as revealed with a mitochondrial in vitro translation system

Takemoto¹,

Spremulli²,

Benkowski³

et al. 2009

Nucleic Acids Research

105

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Mitochondrial (mt) tRNAMet has the unusual modified nucleotide 5-formylcytidine (f5C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNAMet carrying f5C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNAMet lacking f5C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNAMetm with the anticodon ac4CAU (ac4C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNAMet (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f5C modification in mt tRNAMet plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f5C-G and f5C-A based on the chemical properties of f5C are presented.

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Section: Materals and Methodsmentioning

confidence: 99%

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA Met with the anticodon f 5 CAU as revealed with a mitochondrial in vitro translation system

Takemoto¹,

Spremulli²,

Benkowski³

et al. 2009

Nucleic Acids Research

105

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“…14). The remarkable feature of the EF-Tu-GDP complex is that it does not interact with AA-tRNA (3,15,16). C-A-Phe (la), however, interacts with EF-Tu-GDP in a manner similar to its interaction with EF-Tu-GTP as indicated by the Millipore filter technique and gel filtration.…”

Section: Methodsmentioning

confidence: 99%

Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.

Ringer¹,

Chládek²

1975

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

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The interaction between Escherichia coli elongation factor-Tu-GTP complex and chemically synthesized 2'(3')-O-aminoacyldinucleoside phosphates with the nucleotide sequence of the 3' terminus of aminoacyl-tRNA (AA-tRNA) has been studied. It was found that C-A-Phe, C-A-Pro, and C-A-Asp interact with EF-Tu-GTP, causing the release of GTP bound to the enzyme. The specificity of this interaction closely resembles that of AA-tRNA since C-A and C-A(Ac-Phe) as well as the corresponding tRNAs are inactive. The 3'-O-aminoacyl derivative C-2'-dA-Phe does not interact with EF-Tu-GTP, whereas the 2'-O-aminoacyl derivative C-3'-dA-Phe is almost as active as the 2'(3')-O-aminoacyl derivative, C-A-Phe. C-A-Phe also interacts with the EF-Tu-GDP complex in a manner similar to its interaction with EF-Tu-GTP. It is concluded that interaction of 2'(3')-O-aminoacyloligonucleotides possessing the sequence of the 3' terminus of AA-tRNA is analogous to the interaction of that terminus with EF-Tu and it is suggested that EF-Tu is specific for 2'-O-AA-tRNA.

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“…Nitrocellulose binding assays were used to determine inhibition of GDP exchange by EF-Tu as previously described (5,38), with the exception that the enzyme (12.0 M) was preincubated with 0. EF-Tu ternary-complex formation assay.…”

Section: A/t Assaysmentioning

confidence: 99%

Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

Ribble

Hill

Ochsner

et al. 2010

Antimicrob Agents Chemother

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Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyltRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC 50 s) ranging from 3 to 14 M. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five-or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 g/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

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A study of the enzymic transfer of aminoacyl-RNA to Escherichia coli ribosomes

Cited by 89 publications

References 20 publications

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA Met with the anticodon f 5 CAU as revealed with a mitochondrial in vitro translation system

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA Met with the anticodon f 5 CAU as revealed with a mitochondrial in vitro translation system

Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.

Discovery and Analysis of 4 H -Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors

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