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Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3.

Yoo JH, RajBhandary UL - Mol. Microbiol. (2008)

Bottom Line: Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation.In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation.Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV-geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA - formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site - are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.

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Model for translation re-initiation in E. coli. Proposed molecular events occurring between translation termination at the upstream gene (light grey bar) and subsequent re-initiation at the downstream gene (dark grey bar). IF2 enhances re-initiation efficiency, while elevated levels of IF3 decrease it. Our results also suggest that a 30S ribosomal subunit is used for accurate translation re-initiation.
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fig10: Model for translation re-initiation in E. coli. Proposed molecular events occurring between translation termination at the upstream gene (light grey bar) and subsequent re-initiation at the downstream gene (dark grey bar). IF2 enhances re-initiation efficiency, while elevated levels of IF3 decrease it. Our results also suggest that a 30S ribosomal subunit is used for accurate translation re-initiation.

Mentions: We have shown that the di-cistronic CAT-fLuc reporter developed here is an excellent system for studying translation re-initiation. Using anticodon mutants of initiator tRNA (Fig. 1B), we demonstrate that a mutant fLuc reporter gene can be translated by re-initiation from non-AUG initiation codons (Figs 4 and 7). Introduction of additional mutations elsewhere in the initiator tRNA allowed us to identify components of the translation initiation machinery involved in re-initiation. The importance of formylation of the initiator tRNA for its activity in re-initiation indicates that IF2 is required for re-initiation, while a requirement for three consecutive G:C base pairs in the anticodon stem of the initiator tRNA indicates that IF3 also plays a role in re-initiation. Re-initiation appears to have a higher requirement for IF2 than de novo initiation, whereas overproduction of IF3 inhibits re-initiation. Thus, as suggested for leaderless mRNAs, the relative levels of IF2 and IF3 may also influence translation re-initiation efficiency (Fig. 10).


Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3.

Yoo JH, RajBhandary UL - Mol. Microbiol. (2008)

Model for translation re-initiation in E. coli. Proposed molecular events occurring between translation termination at the upstream gene (light grey bar) and subsequent re-initiation at the downstream gene (dark grey bar). IF2 enhances re-initiation efficiency, while elevated levels of IF3 decrease it. Our results also suggest that a 30S ribosomal subunit is used for accurate translation re-initiation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268962&req=5

fig10: Model for translation re-initiation in E. coli. Proposed molecular events occurring between translation termination at the upstream gene (light grey bar) and subsequent re-initiation at the downstream gene (dark grey bar). IF2 enhances re-initiation efficiency, while elevated levels of IF3 decrease it. Our results also suggest that a 30S ribosomal subunit is used for accurate translation re-initiation.
Mentions: We have shown that the di-cistronic CAT-fLuc reporter developed here is an excellent system for studying translation re-initiation. Using anticodon mutants of initiator tRNA (Fig. 1B), we demonstrate that a mutant fLuc reporter gene can be translated by re-initiation from non-AUG initiation codons (Figs 4 and 7). Introduction of additional mutations elsewhere in the initiator tRNA allowed us to identify components of the translation initiation machinery involved in re-initiation. The importance of formylation of the initiator tRNA for its activity in re-initiation indicates that IF2 is required for re-initiation, while a requirement for three consecutive G:C base pairs in the anticodon stem of the initiator tRNA indicates that IF3 also plays a role in re-initiation. Re-initiation appears to have a higher requirement for IF2 than de novo initiation, whereas overproduction of IF3 inhibits re-initiation. Thus, as suggested for leaderless mRNAs, the relative levels of IF2 and IF3 may also influence translation re-initiation efficiency (Fig. 10).

Bottom Line: Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation.In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation.Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV-geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA - formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site - are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.

Show MeSH
Related in: MedlinePlus