New Features of the Ribosome and Ribosomal Inhibitors: Non-Enzymatic Recycling, Misreading and Back-Translocation

Szaflarski, Witold; Vesper, Oliver; Teraoka, Yoshika; Plitta, Beata P.; Wilson, Daniel N.; Nierhaus, Knud H.

doi:10.1016/j.jmb.2008.04.060

Cited by 50 publications

(45 citation statements)

References 42 publications

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“…Under steady-state conditions, such as that seen during poly(U)-dependent poly(Phe) synthesis, the misincorporation of the near-cognate amino acid Leu is Ͻ0.1% (Leu versus Leu plus Phe incorporation) by using our polyamine buffer system (3,30). We analyzed the error of near-cognate dipeptide formation in the presence of a heteropolymeric mRNA with ribosome⅐P-tRNA complexes, containing either a free (Pi) or an occupied E-site (posttranslocational state (POST) with tRNAs at P-and E-sites).…”

Section: Resultsmentioning

confidence: 99%

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

Giacco

Márquez

Qin

et al. 2008

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

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During translation, usually only one in Ϸ400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation. E-site ͉ translational errorsT he binding of aminoacyl-tRNAs to the ribosome is dictated by the complementarity between the anticodon of the tRNA and the codon of the mRNA. To ensure the high fidelity of translation, the correct stereochemistry of the mRNA-tRNA codon-anticodon interaction is monitored by components of the small ribosomal subunit in a process known as decoding (reviewed in ref. 1). During decoding, the first and second nucleotide positions (in terms of the codon) of the mRNA-tRNA duplex are closely monitored, whereas interaction at the third or wobble position is less strictly recognized. Consistently, the misincorporation of the wrong amino acids into polypeptide chains usually occurs through the binding of nearcognate aminoacyl-tRNAs, i.e., those tRNAs carrying an anticodon similar to that of the cognate aminoacyl-tRNA, rather than noncognate aminoacyl-tRNAs, which carry dissimilar anticodons. Nascent polypeptide chains are surprisingly tolerant to misincorporation, with only one in Ϸ400 misincorporations being deleterious for the protein's activity (reviewed in ref.2). The reason for this is that usually near-cognate aminoacyl-tRNAs are selected instead of the cognate aminoacyl-tRNA, and the genetic code lexicon is organized in such a way that near-cognate tRNAs bear amino acids that are chemically similar to those carried by the cognate tRNA. For example, the misincorporation of an aspartate (codon: GAU/C) by near-cognate Asp-tRNA, instead of glutamate (GAA/G) by the cognate Glu-tRNA, both incorporate acidic amino acids. The middle and, in most cases, the first position of a codon are almost never misread, even under error-inducing conditions such as high magnesiu...

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Section: Resultsmentioning

confidence: 99%

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

Giacco

Márquez

Qin

et al. 2008

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

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“…1). These three C-5Љ-dihydro compounds and compound 7 were studied for their ability both to inhibit the growth of ⌬tolC E. coli and to inhibit protein synthesis in an E. coli in vitro-coupled transcriptiontranslation system (5,30). The MICs of DHHA, compound 2, and compound 4 were determined to be 20 g/ml, 150 g/ml, and 150 g/ml, respectively (Table 3).…”

Section: Resultsmentioning

confidence: 99%

“…All coupled transcription-translation experiments were performed with an E. coli lysate-based system in the presence and absence of antibiotics, as described previously (5,30). The reaction mixtures were transferred into 96-well microtiter plates, and the fluorescence of the green fluorescent protein (GFP) was measured with a Typhoon 9400 scanner (Amersham Bioscience) and a Typhoon blue laser module (Amersham Bioscience).…”

Section: Methodsmentioning

confidence: 99%

The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

Dhote

Starosta

Wilson

et al. 2009

Antimicrob Agents Chemother

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Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5؆-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in Escherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the ⌬hyg19 or the ⌬hyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.

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“…In a system which contains only Mg 2ϩ and monovalent cations such as K ϩ or NH 4 ϩ , the ribosomes will become inactivated at Mg 2ϩ concentrations below 10 mM; one has to add polyamines, most importantly spermidine (2 mM), in order to rescue the activity and allow the protein synthesis rate even to approach in vivo perfection (11)(12)(13)(14)(15). The optimized systems contain 2 to 5 mM Mg 2ϩ .…”

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confidence: 99%

Mg ²⁺ , K ⁺ , and the Ribosome

Nierhaus

2014

J Bacteriol

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101

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New Features of the Ribosome and Ribosomal Inhibitors: Non-Enzymatic Recycling, Misreading and Back-Translocation

Cited by 50 publications

References 42 publications

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

Mg ²⁺ , K ⁺ , and the Ribosome

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New Features of the Ribosome and Ribosomal Inhibitors: Non-Enzymatic Recycling, Misreading and Back-Translocation

Cited by 50 publications

References 42 publications

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux

Mg 2+ , K + , and the Ribosome

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Mg ²⁺ , K ⁺ , and the Ribosome