The use of nuclease P<sub>1</sub>in sequence analysis of end group labeled RNA

Silberklang, Melvin; Gillum, Amanda M.; RajBhandary, Uttam L.

doi:10.1093/nar/4.12.4091

Cited by 249 publications

(109 citation statements)

References 21 publications

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“…3¢-32 P-labeling of tRNA Phe was performed with tRNA nucleotidyltransferase according to [20]. The labeled tRNA was purified in 8% polyacrylamide gel containing 8 M urea.…”

Section: Trnamentioning

confidence: 99%

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Petrushenko

Budkevich

Shalak

et al. 2002

European Journal of Biochemistry

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Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl‐tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13–17]. The [eEF1A·GDP·tRNA] complex has been hypothesized to interact with aminoacyl‐tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A·GDP·tRNA] complex with phenylalanyl‐tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel‐retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A·GDP·tRNA] and [eEF1A·GDP·tRNAPhe·PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A·GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A·GDP with tRNA and ARS in the channeled elongation cycle is discussed.

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“…3¢-32 P-labeling of tRNA Phe was performed with tRNA nucleotidyltransferase according to [20]. The labeled tRNA was purified in 8% polyacrylamide gel containing 8 M urea.…”

Section: Trnamentioning

confidence: 99%

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Petrushenko

Budkevich

Shalak

et al. 2002

European Journal of Biochemistry

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“…Labeling at the 5'-end of the RNAs is performed with [γ-32 P]-ATP and phage T4 polynucleotide kinase after dephosphorylation with alkaline phosphatase [10]. Labeling at the 3'-end is done by ligation of [γ-…”

Section: Rna Labelingmentioning

confidence: 99%

Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA

Bordeau¹,

Felden²

2002

Biochimie

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To cite this version:Valérie Bordeau, Brice Felden. Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA.. Biochimie, Elsevier, 2002, 84 (8) Abstract tmRNA (10Sa RNA, ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. Ribosomal protein S1 is required for tmRNA binding to isolated and poly U-programmed ribosomes. Mobility assays on native gels indicate that the binding curves of both recombinant and purified proteins S1 from E. coli is biphasic with apparent binding constants of ∼90 and ∼300 nM, respectively, suggesting that more than one protein interacts with tmRNA. Structural probing of native tmRNA in the presence and absence of the purified protein suggest that when S1 binds, tmRNA undergoes a significant conformational change. In the presence of the protein, nucleotides from tmRNA with enhanced (H2, H3, PK1, PK2, PK4, in and around the first triplet to be translated), or decreased (H5 and PK2), reactivity towards a probe specific for RNA single-strands are scattered throughout the molecule, with the exception of the tRNA-like domain that may be dispensable for the interaction. Converging experimental evidence suggests that ribosomal protein S1 binds to pseudoknot PK2. Previous structural studies of tmRNA in solution have revealed several discrepancies between the probing data and the phylogeny, and most of these are reconciled when analyzing tmRNA structure in complex with the protein(s). Ribosomal protein(s) S1 is proposed to set tmRNA in the mRNA mode, relieving strains that may develop when translating a looped mRNA.

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“…In addition to the RNases Ti and U2 the enzymes Bc (22), C13 (23,24) and PhyM (25) were used. In Coleosporium tussilaginis the 5'-endgroup of the 5S rRNA molecule was confirmed by mobility shift analysis (26). Sequencing gels were 12%, 15% and 20% polyacrylamide, 8.3 M urea.…”

Section: Methodsmentioning

confidence: 93%

Highly conserved 5S ribosomal RNA sequences in four rust fungi and atypical 5S rRNA secondary structure inMicrostroma juglandis

Gottschalk

Blanz

1984

Nucl Acids Res

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The 5S ribosomal RNA nucleotide sequences of five basidiomycetous fungi, Coleosporium tussilaginis, Gymnosporangium clavariaeforme, Puccinia poarum, Endophyllum sempervivi and Microstroma juglandis were determined.Despite high differentiation in their host spectra the four rust species are highly conserved with respect to their 5S rRNA sequences, which fit with the basidiomycete cluster 5 described by Walker and Doolittle (1).The sequences obtained from the first three rust fungi were proven to be identical while the sequence from Endophyllum sempervivi showed two base substitutions compared with the other rust fungi. The Microstroma juglandis 5S rRNA sequence differs from all other basidiomycete 5S rRNA sequences published so far in respect to its secondary structure which shows an atypical 'CCA' loop in helix D, but it reveals typical basidiomycetous signature nucleotides. Therefore Microstroma juglandis represents a cluster of its own within the Basidiomycetes. A dendrogram was constructed based on Kimura's "Neutral Theory of Molecular Evolution".

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The use of nuclease P₁in sequence analysis of end group labeled RNA

Cited by 249 publications

References 21 publications

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA

Highly conserved 5S ribosomal RNA sequences in four rust fungi and atypical 5S rRNA secondary structure inMicrostroma juglandis

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The use of nuclease P1in sequence analysis of end group labeled RNA

Cited by 249 publications

References 21 publications

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA

Highly conserved 5S ribosomal RNA sequences in four rust fungi and atypical 5S rRNA secondary structure inMicrostroma juglandis

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The use of nuclease P₁in sequence analysis of end group labeled RNA