Effective non‐viral leader for cap‐independent translation in a eukaryotic cell‐free system

Shaloiko, Lyubov; Granovsky, Igor; Ивашина, Т. В.; Ksenzenko, V. N.; Широков, В. Б.; Spirin, Alexander S.

doi:10.1002/bit.20267

Cited by 17 publications

(13 citation statements)

References 54 publications

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“…This value was in agreement with the concentration of the limiting initiation factor eIF4F. Further addition of mRNA caused inhibition of complex assembly that can be explained by the partial sequestration of protein factors by excess mRNA [mRNA self inhibition effect, see Refs (11,13)]. At the same time, at very low mRNA concentrations, some impurities like cross-contamination of the factors may have a significant effect on complex formation.…”

Section: Resultssupporting

confidence: 72%

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Shirokikh

Alkalaeva

Vassilenko

et al. 2009

Nucleic Acids Research

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Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.

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Section: Resultssupporting

confidence: 72%

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Shirokikh

Alkalaeva

Vassilenko

et al. 2009

Nucleic Acids Research

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“…16, final total protein concentration 3 mg/ml) was carried out at 30°C in the presence of 26.1 mM Hepes KOH, pH 7.6, 2% glycerol, 1.6 mM DTT, 0.05 g/liter of bulk yeast tRNA, 0.1 mM luciferin, 16 mM creatine phosphate, 0.4 mM GTP, UTP, CTP, 1 mM ATP, 3 mM Mg(OAc) 2 , 70 mM KOAc, 0.2 mM each amino acid, 0.45 mM spermidine, 74 nM luciferase mRNA, and 0.1 g/liter of creatine kinase. The reaction progression was monitored as above.…”

Section: Preparation Of Cell Extract For MCC Processing-mcc-sensitivementioning

confidence: 99%

Aspartyl-tRNA Synthetase Is the Target of Peptide Nucleotide Antibiotic Microcin C

Metlitskaya

Kazakov

Kommer

et al. 2006

Journal of Biological Chemistry

142

157

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Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.Genes responsible for microcin production are usually plasmidborne. Plasmids encoding microcin structural and maturation genes also encode determinants of immunity specific to the microcin produced. Based on cross-immunity, post-translationally modified microcins can be subdivided into the B, C, and J types. Microcin B (MccB) 4 is a 43-residue peptide with 8 thiazole and oxazole rings that are synthesized by the McbBCD maturation enzyme complex from multiple serine and cysteine residues present in the MccB precursor (4). MccB is a potent inhibitor of DNA gyrase; it traps the enzyme at the stage of DNA strand passage (5). Microcin J, a 21-amino acid peptide, contains an unusual lactam bond between its N-terminal glycine and the ␦-carboxyl group of an internal glutamate; it assumes a highly unusual threaded-lasso structure (6 -8). MccJ inhibits bacterial RNA polymerase by occluding a narrow channel that is used to traffic transcription substrates, NTPs, to the catalytic center of the enzyme (9, 10).The structure of the subject of this study, Microcin C (McC) is shown in Fig. 1A. McC is a heptapeptide containing a modified adenosine monophosphate covalently attached to its C terminus through an N-acylphosphoramidate linkage (11, 12). The phosphoramidate group of the nucleotide part of McC is additionally modified by a propylamine group. Additionally, in mature McC, the peptide moiety, which is encoded by the mccA gene, is modified and the C-terminal asparagine residue specified by mccA is converted to an aspartate (18, 19), through an unknown mechanism. In vivo, McC appears to target translation (12). Guijarro et al. (12) also reported that large concentrations of McC, as well as of synthetic peptide of the same sequence but without the nucleotide modification, mildly inhibit translation in vitro. They therefore concluded that the peptide part of McC is responsible for transl...

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“…However, the cap stimulates translation only at lower mRNA concentrations (within the range up to 200 pmol/mL). At the same time, cap is found inhibitory at high concentrations mRNA (at 400 pmol/mL and higher) due to the inhibition of translation by excess mRNA (the so-called self-inhibition effect; see Shaloiko et al, 2004). Uncapped construct with omega leader also displays self-inhibition.…”

Section: Cell-free Translation In Batch Formatmentioning

confidence: 99%

5′‐poly(A) sequence as an effective leader for translation in eukaryotic cell‐free systems

Gudkov

Ozerova

Shiryaev

et al. 2005

Biotech & Bioengineering

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Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.

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Effective non‐viral leader for cap‐independent translation in a eukaryotic cell‐free system

Cited by 17 publications

References 54 publications

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Aspartyl-tRNA Synthetase Is the Target of Peptide Nucleotide Antibiotic Microcin C

5′‐poly(A) sequence as an effective leader for translation in eukaryotic cell‐free systems

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