Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

Grill, Sonja; Moll, Isabella; Hasenöhrl, David; Gualerzi, Claudio O.; Bläsi, Udo

doi:10.1016/s0014-5793(01)02378-x

Cited by 47 publications

(42 citation statements)

References 30 publications

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“…Moreover, as S1 is widely conserved and plays a role in translation initiation in distantly related bacteria, [8][9][10] it seems sound that molecules targeting the S1-dependent mechanism identified in E. coli may inhibit translation and thus the growth of a large spectrum of bacteria. Another advantage of our assay is that, since only a few cellular factors seem to be specifically involved in leadered versus leaderless translation initiation, 12,32 the determination of the molecular target(s) of the hits should be easier than with other phenotypic screenings.…”

Section: Discussionmentioning

confidence: 99%

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

2015

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The bacterial translational apparatus is an ideal target for the search of new antibiotics. In fact, it performs an essential process carried out by a large number of potential subtargets for antibiotic action. Moreover, it is sufficiently different in several molecular details from the apparatus of Eukarya and Archaea to generally ensure specificity for the bacterial domain. This applies in particular to translation initiation, which is the most different step in the process. In bacteria, the 30S ribosomal subunit directly binds to the translation initiation region, a site within the messenger RNA (mRNA) 5′-untranslated region (5′-UTR). 30S binding is mediated by the interaction of both the 16S ribosomal RNA and the ribosomal protein S1 with specific regions of the mRNA 5′-UTR. An alternative, S1-independent pathway is enjoyed by leaderless mRNAs (i.e., transcripts devoid of a 5′-UTR). We have developed a simple fluorescence-based whole-cell assay in Escherichia coli to find inhibitors of the canonical S1-dependent translation initiation pathway. The assay has been set up both in a common E. coli laboratory strain and in a strain with an outer membrane permeability defect. Compared with other whole-cell assays for antibacterials, the major advantages of the screen described here are high sensitivity and specificity.

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Section: Discussionmentioning

confidence: 99%

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

2015

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“…An increasing number of non-SD-led genes (Chang et al 2006), including genes encoding leaderless mRNA completely lacking a 59 untranslated region (UTR) (Janssen 1993;Janssen 1996, 1997;Tedin et al 1997;Grill et al 2000Grill et al , 2001Moll et al 2002; and references therein), continue to be identified. Leaderless mRNAs have been reported to follow a novel pathway of translation initiation involving 70S ribosomes (Balakin et al 1992;O'Donnell and Janssen 2002;Moll et al 2004;Udagawa et al 2004) or 30S subunits free of IF3, which is reported to discriminate against a 59-terminal start codon (Moll et al 1998;Tedin et al 1999;Grill et al 2000Grill et al , 2001.…”

Section: Introductionmentioning

confidence: 99%

“…Leaderless mRNAs have been reported to follow a novel pathway of translation initiation involving 70S ribosomes (Balakin et al 1992;O'Donnell and Janssen 2002;Moll et al 2004;Udagawa et al 2004) or 30S subunits free of IF3, which is reported to discriminate against a 59-terminal start codon (Moll et al 1998;Tedin et al 1999;Grill et al 2000Grill et al , 2001. Toeprint assays have demonstrated tRNA-dependent ribosome binding to leaderless mRNA, and an AUG start codon is required for efficient ribosome binding and translation in Escherichia coli (Van Etten and Janssen 1998;O'Donnell and Janssen 2001).…”

Section: Introductionmentioning

confidence: 99%

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5′-terminal AUG

Brock

Pourshahian²,

Giliberti³

et al. 2008

RNA

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Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage l, with a 4-thiouridine (4 S -U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 59-AUG is required for stable binding. Furthermore, the addition of a 59-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.

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“…Although the directed evolution of ribosomal protein S1 improves the translational efficiency of expression from these foreign DNA sequences, we recognize that other proteins and perhaps also the RNA core of the ribosome are likely to influence the expression of foreign DNA in the cell. Other proteins such as translation initiation factors IF1, IF2, and IF3 certainly play a role in translation initiation (11,39,40), and we cannot rule out the possibility of accessory proteins in E. coli or from a donor organism that may be used to stabilize mRNAs or prepare them for efficient translation. We have considered that many high GC bacteria have optimal growth rates near 30°C, yet they have evolved to express their high GC mRNAs at temperatures that would allow for extensive formation of secondary structures within transcripts.…”

Section: Genementioning

confidence: 99%

Directed Evolution of Ribosomal Protein S1 for Enhanced Translational Efficiency of High GC Rhodopseudomonas palustris DNA in Escherichia coli

Bernstein

Bülter

Shen

et al. 2007

Journal of Biological Chemistry

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The expression of foreign DNA in Escherichia coli is important in biotechnological applications. However, the translation of genes from GC-rich organisms is inefficient in E. coli. To overcome this problem, we applied directed evolution to E. coli ribosomal protein S1. Two selected mutants enabled 12-and 8-fold higher expression levels from GC-rich DNA targets. General improvements in translation efficiency over a range of genes from Rhodopseudomonas palustris and E. coli was achieved using an S1 mutant selected against multiple genes from R. palustris. This method opens new opportunities for the expression of GC-rich genes in E. coli.The recombinant expression of DNA in Escherichia coli is important for biotechnological applications, enabling the extension of pathways for the production of non-native metabolites with a growing economic and social impact (1-5). Heterologous expression of GC-rich genes in E. coli poses a unique set of challenges, in comparison with other DNA. Overcoming these problems will generate increased flexibility in the production of proteins and metabolites in this host. Although most efforts for recombinant expression in this host are achieved using E. coli promoters and optimal ribosome-binding sites (RBS), 2 it would be advantageous to express large pathways, consisting of many genes, without needing to change each promoter and RBS. Such a strategy would enable hybrid functionalities from multiple organisms using technology such as the fusing of two genomes (6) or transformation-associated recombination (7). Here we assess the ability of E. coli to express native DNA, including transcription and translation initiation regions, from the high GC ␣-bacterium Rhodopseudomonas palustris (8), and we propose a novel method for enhancing expression of foreign transcripts, altering E. coli ribosomal protein S1 by directed evolution.

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Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

Cited by 47 publications

References 30 publications

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

A Whole-Cell Assay for Specific Inhibitors of Translation Initiation in Bacteria

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5′-terminal AUG

Directed Evolution of Ribosomal Protein S1 for Enhanced Translational Efficiency of High GC Rhodopseudomonas palustris DNA in Escherichia coli

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