Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits

Favaudon, Vincent; François, Patrice

doi:10.1021/bi00663a001

Cited by 10 publications

(4 citation statements)

References 37 publications

(29 reference statements)

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“…Because efficient extraction of membrane-bound polysomes requires detergent (Potter and Nicchitta, 2002), we expected cytoplasmic species to predominate in our lysates. Although Mg 2+ concentrations up to 30 mM are often used when preparing yeast extract for polysome profiling (Bhattacharya et al, 2010), [Mg 2+ ] in excess of 8 mM can drive vacant couple formation (Favaudon and Pochon, 1976); therefore, we limited total [Mg 2+ ] to 5 mM in all experiments.…”

Section: Monosome Polysome and Global Footprintingmentioning

confidence: 99%

Redefining the Translational Status of 80S Monosomes

Heyer

Moore

2016

Cell

194

181

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Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remain controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ∼ 590 nt, and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.

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Section: Monosome Polysome and Global Footprintingmentioning

confidence: 99%

Redefining the Translational Status of 80S Monosomes

Heyer

Moore

2016

Cell

194

181

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“…It also reduces the electrostatic repulsion between the subunits by masking the charges on the rRNA [25]. In both prokaryotes and mitochondria, spermine has been shown to tighten the interaction between the ribosomal subunits [15,26].…”

Section: Resultsmentioning

confidence: 99%

The effect of spermine on the initiation of mitochondrial protein synthesis

Christian¹,

Haque²,

Spremulli³

2010

Biochemical and Biophysical Research Communications

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Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2 mt ) and mitochondrial initiation factor 3 (IF3 mt ). This observation indicates that IF2 mt and IF3 mt , while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3 mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2 mt . The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

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“…It has been reported that the equilibrium between 70 S ribosomes and the 30 S and 50 S subunits is influenced by polyamines (21,22). In the presence of 2-3 mM Mg 2ϩ and 100 mM K ϩ , however, the percentage of 70 S ribosomes (70 -80% of total ribosomes) was not influenced strongly by 0.5-1 mM spermidine (data not shown).…”

Section: Determination Of the Size Of Oppa Mrna Is Necessary For The mentioning

confidence: 99%

Polyamine Stimulation of the Synthesis of Oligopeptide-binding Protein (OppA)

Yoshida

Meksuriyen

Kashiwagi

et al. 1999

Journal of Biological Chemistry

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We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T 1 , specific for single-stranded RNA, and RNase V 1 , which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMettRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.Polyamines (putrescine, spermidine, and spermine) are necessary for normal cell growth, and their proliferative effects are probably due to stimulation of nucleic acid and protein synthesis (1, 2). We previously reported that polyamines can stimulate the synthesis of some types of prokaryotic and eukaryotic proteins in cell-free systems (3, 4) and in vivo (5, 6) and that the assembly of 30 S ribosomal subunits is stimulated by polyamines (7, 8). We also found that most polyamines exist as a polyamine-RNA complex in cells (9). In a rabbit reticulocyte cell-free system, under conditions in which spermidine produced a maximal stimulation of globin synthesis, the amount of polyamine bound to RNA was very close to the value estimated for intact cells, suggesting an important physiological role for polyamines bound to RNA (10).In Escherichia coli, the synthesis of a protein termed PI 1 (polyamine-induced) protein was strongly stimulated by the addition of putrescine to a polyamine-requiring mutant MA261 (11). The PI protein was identified, by cloning of its gene, as OppA, which is a periplasmic substrate-binding protein of the oligopeptide uptake system (12). We found that stimulation of OppA synthesis by polyamines occurs at the level of translation and that the position and secondary structure of the ShineDalgarno (SD) sequence (13) are probably important for stimulation by polyamines (14). In the present work we wanted to clarify the nature of the structural change of OppA mRNA induced by polyamines that is responsible for stimulation of OppA protein synthesis. For this purpose, four different 130-mer mRNAs containing the initiation region of OppA were synthesized in vitro and used to study both the structu...

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Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits

Cited by 10 publications

References 37 publications

Redefining the Translational Status of 80S Monosomes

Redefining the Translational Status of 80S Monosomes

The effect of spermine on the initiation of mitochondrial protein synthesis

Polyamine Stimulation of the Synthesis of Oligopeptide-binding Protein (OppA)

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