Ribosome reactivation by replacement of damaged proteins

Pulk, Arto; Liiv, Aivar; Peil, Lauri; Maiväli, Ülo; Nierhaus, Knud H.; Rèmme, Jaanus

doi:10.1111/j.1365-2958.2009.07002.x

Cited by 65 publications

(63 citation statements)

References 62 publications

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“…Nevertheless, data presented in this work provide a new tool for the isolation and further characterization of E. coli ribosomal precursor particles, and more generally for future structure-function relationship studies of the bacterial ribosome. These precursor particles can be reactivated in vivo (Figure 2B) and in vitro (data not shown), probably by a process of protein replacement that was recently described in the reactivation of stationary phase ribosomes (51). …”

Section: Discussionmentioning

confidence: 57%

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

René

Alix

2010

Nucleic Acids Research

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The late stages of 30S and 50S ribosomal subunits biogenesis have been studied in a wild-type (wt) strain of Escherichia coli (MC4100) subjected to a severe heat stress (45–46°C). The 32S and 45S ribosomal particles (precursors to 50S subunits) and 21S ribosomal particles (precursors to 30S subunits) accumulate under these conditions. They are authentic precursors, not degraded or dead-end particles. The 21S particles are shown, by way of a modified 3′5′ RACE procedure, to contain 16S rRNA unprocessed, or processed at its 5′ end, and not at the 3′ end. This implies that maturation of 16S rRNA is ordered and starts at its 5′-terminus, and that the 3′-terminus is trimmed at a later step. This observation is not limited to heat stress conditions, but it also can be verified in bacteria growing at a normal temperature (30°C), supporting the idea that this is the general pathway. Assembly defects at very high temperature are partially compensated by plasmid-driven overexpression of the DnaK/DnaJ chaperones. The ribosome assembly pattern in wt bacteria under a severe heat stress is therefore reminiscent of that observed at lower temperatures in E. coli mutants lacking the chaperones DnaK or DnaJ.

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Section: Discussionmentioning

confidence: 57%

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

René

Alix

2010

Nucleic Acids Research

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“…Protein S1 is known to affect mRNA binding to the ribosome (16). As binding of S1 to the small ribosome subunit is labile and is easily exchangeable (17), small changes such as those seen here may not be significant in general. However, given that the initiation activities of the elongator tRNAs are also low, we considered that such a small change in S1 on ribosomes, occurring as a consequence of deletion of metZWV, may contribute to the initiation activity of the elongator tRNAs.…”

Section: Figmentioning

confidence: 82%

Initiation with Elongator tRNAs

Samhita

Virumäe

Rèmme

et al. 2013

Journal of Bacteriology

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In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution. Most organisms possess two kinds of tRNAs, the initiator tRNA, which decodes the initiation codon, and the elongator tRNAs, which decode the subsequent codons within the open reading frame (ORF). In Escherichia coli, there are four copies of the initiator tRNA genes; three of these, metZ, metW, and metV, are present at 63.5 min, whereas the fourth one, metY, is found at 71.5 min in the genome (1). It is also known that either (but not both) of these loci may be deleted from E. coli. The strain deleted for metZWV becomes cold sensitive. However, at temperatures of 30°C or higher the strain grows normally (2, 3).Initiator and elongator tRNAs are believed to perform their respective decoding functions without functional interference from one another. Although they share the same cloverleaf-like backbone structure, years of evolution have led to their separate and distinct functions, now held in place by an intricate support system comprising translation factors, quality control mechanisms, and the ribosome itself. Elongator tRNAs are excluded from participation at the start of protein synthesis by sequence features specific to the initiator tRNA that are recognized exclusively at the P site of the ribosome (4-6). Various other details, such as the fact that the initiation and elongation factors bind specifically to their cognate species of tRNAs, further aid the specificity of their functions.However, earlier studies have enabled initiation by elongator tRNAs, by transplanting the critical features of initiator tRNA onto the...

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“…The modification sites that are buried deep inside the subunit must be modified during early assembly. Finally, it is noteworthy that a specific set of r-proteins are exchangeable in vivo (Pulk et al 2010). When the ribosome bound r-proteins can be exchanged with the free r-proteins, they could be displaced by the rRNA modifications as well.…”

Section: Discussionmentioning

confidence: 99%

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Siibak¹,

Rèmme²

2010

RNA

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Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

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Ribosome reactivation by replacement of damaged proteins

Cited by 65 publications

References 62 publications

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

Initiation with Elongator tRNAs

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

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