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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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The fLuc reporter gene is translated by re-initiating ribosomes. A. Relative fLuc activity from mutant di-cistronic reporters containing premature UAG stop codons placed at various distances upstream of the normal geneV UAA stop codon. SupF suppressor or U35A36 mutant initiator tRNA was also coexpressed. fLuc activity from CL was set at 100%. nt, number of nucleotides separating the two reporter genes. fLuc activity is defined as RLU/OD600. B. Immunoblot of total-cell extract from (A) using anti-CAT or anti-fLuc Ab. The mutant di-cistronic reporter containing a 40-nucleotide intercistronic region (Cam219L, lane 4) encodes a CAT protein that is ∼1.4 kDa smaller than the full-length CAT. Lane 6 represents a sample from E. coli transformed with empty vector. C. Relative fLuc activity from mutant di-cistronic reporters without or with a SD sequence upstream of the fLuc start codon (CrbsL). fLuc activity from CL was set at 1. fLuc activity is defined as RLU/OD600.
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fig03: The fLuc reporter gene is translated by re-initiating ribosomes. A. Relative fLuc activity from mutant di-cistronic reporters containing premature UAG stop codons placed at various distances upstream of the normal geneV UAA stop codon. SupF suppressor or U35A36 mutant initiator tRNA was also coexpressed. fLuc activity from CL was set at 100%. nt, number of nucleotides separating the two reporter genes. fLuc activity is defined as RLU/OD600. B. Immunoblot of total-cell extract from (A) using anti-CAT or anti-fLuc Ab. The mutant di-cistronic reporter containing a 40-nucleotide intercistronic region (Cam219L, lane 4) encodes a CAT protein that is ∼1.4 kDa smaller than the full-length CAT. Lane 6 represents a sample from E. coli transformed with empty vector. C. Relative fLuc activity from mutant di-cistronic reporters without or with a SD sequence upstream of the fLuc start codon (CrbsL). fLuc activity from CL was set at 1. fLuc activity is defined as RLU/OD600.

Mentions: The di-cistronic reporter system was characterized to confirm that both reporters, CAT and fLuc, were co-transcribed and co-translated. E. coli CA274 cells were transformed with the wild-type di-cistronic reporter CL, induced with arabinose, and cell extracts analysed for CAT and fLuc activity, and for protein expression levels using immunoblot analysis. Assays for fLuc activity showed a parallel increase in activity with increasing levels of arabinose (Fig. 2A, graph). Immunoblot analyses revealed that expression of both reporters increased in a similar manner with increasing levels of arabinose (Fig. 2A, immunoblot), consistent with both genes being co-transcribed and co-translated. Several faster migrating bands representing internally initiated luciferase fragments were also detected with the anti-fLuc antibody (Ab). These bands do not represent degradation products derived from full-length fLuc and contribute little, if any, to fLuc activity (compare levels of truncated fragments in Fig. 3B, lane 4, with their activity in Fig. 3A).


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)

The fLuc reporter gene is translated by re-initiating ribosomes. A. Relative fLuc activity from mutant di-cistronic reporters containing premature UAG stop codons placed at various distances upstream of the normal geneV UAA stop codon. SupF suppressor or U35A36 mutant initiator tRNA was also coexpressed. fLuc activity from CL was set at 100%. nt, number of nucleotides separating the two reporter genes. fLuc activity is defined as RLU/OD600. B. Immunoblot of total-cell extract from (A) using anti-CAT or anti-fLuc Ab. The mutant di-cistronic reporter containing a 40-nucleotide intercistronic region (Cam219L, lane 4) encodes a CAT protein that is ∼1.4 kDa smaller than the full-length CAT. Lane 6 represents a sample from E. coli transformed with empty vector. C. Relative fLuc activity from mutant di-cistronic reporters without or with a SD sequence upstream of the fLuc start codon (CrbsL). fLuc activity from CL was set at 1. fLuc activity is defined as RLU/OD600.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2268962&req=5

fig03: The fLuc reporter gene is translated by re-initiating ribosomes. A. Relative fLuc activity from mutant di-cistronic reporters containing premature UAG stop codons placed at various distances upstream of the normal geneV UAA stop codon. SupF suppressor or U35A36 mutant initiator tRNA was also coexpressed. fLuc activity from CL was set at 100%. nt, number of nucleotides separating the two reporter genes. fLuc activity is defined as RLU/OD600. B. Immunoblot of total-cell extract from (A) using anti-CAT or anti-fLuc Ab. The mutant di-cistronic reporter containing a 40-nucleotide intercistronic region (Cam219L, lane 4) encodes a CAT protein that is ∼1.4 kDa smaller than the full-length CAT. Lane 6 represents a sample from E. coli transformed with empty vector. C. Relative fLuc activity from mutant di-cistronic reporters without or with a SD sequence upstream of the fLuc start codon (CrbsL). fLuc activity from CL was set at 1. fLuc activity is defined as RLU/OD600.
Mentions: The di-cistronic reporter system was characterized to confirm that both reporters, CAT and fLuc, were co-transcribed and co-translated. E. coli CA274 cells were transformed with the wild-type di-cistronic reporter CL, induced with arabinose, and cell extracts analysed for CAT and fLuc activity, and for protein expression levels using immunoblot analysis. Assays for fLuc activity showed a parallel increase in activity with increasing levels of arabinose (Fig. 2A, graph). Immunoblot analyses revealed that expression of both reporters increased in a similar manner with increasing levels of arabinose (Fig. 2A, immunoblot), consistent with both genes being co-transcribed and co-translated. Several faster migrating bands representing internally initiated luciferase fragments were also detected with the anti-fLuc antibody (Ab). These bands do not represent degradation products derived from full-length fLuc and contribute little, if any, to fLuc activity (compare levels of truncated fragments in Fig. 3B, lane 4, with their activity in Fig. 3A).

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