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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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De novo and re-initiation of di-cistronic reporters using UAG as the initiation codon. A. Relative enzyme activity from E. coli CA274 transformed with CL or mutant di-cistronic reporters containing either a mutant CAT reporter (Cam1L) or a mutant fLuc reporter (CLam1) and expressing the U35A36 mutant initiator tRNA. Cell extracts were analysed for CAT (Cam1L, white bar) or fLuc activity (CLam1, grey bar) and compared with activity from CL. CAT activity was determined as described in Experimental procedures and normalized to total protein and β-lactamase activity (to normalize for plasmid copy number). fLuc activity was determined as described in Experimental procedures and normalized to cell number and CAT activity. B. Immunoblots of cell extracts from (A), using anti-CAT (top) or anti-fLuc (bottom) Ab. Different relative amounts of cell extract were analysed to facilitate comparison of protein levels between wild-type and mutant samples.
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fig04: De novo and re-initiation of di-cistronic reporters using UAG as the initiation codon. A. Relative enzyme activity from E. coli CA274 transformed with CL or mutant di-cistronic reporters containing either a mutant CAT reporter (Cam1L) or a mutant fLuc reporter (CLam1) and expressing the U35A36 mutant initiator tRNA. Cell extracts were analysed for CAT (Cam1L, white bar) or fLuc activity (CLam1, grey bar) and compared with activity from CL. CAT activity was determined as described in Experimental procedures and normalized to total protein and β-lactamase activity (to normalize for plasmid copy number). fLuc activity was determined as described in Experimental procedures and normalized to cell number and CAT activity. B. Immunoblots of cell extracts from (A), using anti-CAT (top) or anti-fLuc (bottom) Ab. Different relative amounts of cell extract were analysed to facilitate comparison of protein levels between wild-type and mutant samples.

Mentions: Changing the CAU anticodon of wild-type initiator tRNA to CUA (U35A36 mutant) or GAC (G34C36 mutant) allows the mutant initiator tRNA to initiate from UAG and GUC codons, respectively, in E. coli (Fig. 1B) (Varshney and RajBhandary, 1990; Wu and RajBhandary, 1997). The corresponding G34C36 mutant initiator tRNA can, similarly, be used to initiate de novo protein synthesis from GUC codons in a mutant CAT gene in mammalian cells (Drabkin and RajBhandary, 1998) and in a mutant leaderless bacterio-opsin gene in the archaeon Halobacterium salinarum (Srinivasan et al., 2006). Prior to testing if UAG could also be used as a start codon for translation re-initiation, we confirmed that the mono-cistronic reporter Lam1 could be initiated from UAG by de novo initiation (∼100% efficiency, see Fig. S2). We proceeded to compare the efficiency of the U35A36 mutant initiator tRNA in de novo initiation of mutant CAT from Cam1L and re-initiation of mutant fLuc from CLam1. Based on enzyme activity, the mutant CAT reporter was translated to ∼60% efficiency, while the mutant fLuc reporter was translated to ∼7% efficiency (Fig. 4A) (the efficiencies were calculated relative to enzyme activity from the wild-type reporter, CL). We noticed, however, that there was a discrepancy between mutant CAT enzyme activity and protein levels. Immunoblot analysis showed an almost fivefold reduction in CAT protein levels in cells expressing the Cam1L reporter (Fig. 4B, top) compared with those expressing the wild-type CL reporter, instead of the ∼1.6-fold reduction expected on the basis of CAT activities (Fig. 4A). These results suggest that the specific activity of the mutant CAT protein initiated with formylglutamine and extended at the C-terminus by 13 amino acids, is higher by a factor of ∼3 than wild-type CAT protein initiated with formylmethionine, leading to an overestimation of CAT expression based on enzyme activity. To correct for this inconsistency, we used immunoblotting and densitometric analysis of the immunoblots to calculate the translation initiation efficiencies for mutant CAT expression (Table S1). Use of this assay revealed that the U35A36 mutant initiator tRNA was still more efficient in de novo initiation of mutant CAT (17%) than in re-initiation of the mutant fLuc reporter (7%). The specific activity of the fLuc reporter was not affected by the type of amino acid present at the N-terminus (compare Fig. 4A and B, bottom). Interestingly, a non-fusion, native CAT reporter previously used in our laboratory did not exhibit differences in specific activity when initiating with amino acids other than methionine (Varshney and RajBhandary, 1990; Mangroo and RajBhandary, 1995; Mayer et al., 2003). This was also verified experimentally during this work (data not shown).


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)

De novo and re-initiation of di-cistronic reporters using UAG as the initiation codon. A. Relative enzyme activity from E. coli CA274 transformed with CL or mutant di-cistronic reporters containing either a mutant CAT reporter (Cam1L) or a mutant fLuc reporter (CLam1) and expressing the U35A36 mutant initiator tRNA. Cell extracts were analysed for CAT (Cam1L, white bar) or fLuc activity (CLam1, grey bar) and compared with activity from CL. CAT activity was determined as described in Experimental procedures and normalized to total protein and β-lactamase activity (to normalize for plasmid copy number). fLuc activity was determined as described in Experimental procedures and normalized to cell number and CAT activity. B. Immunoblots of cell extracts from (A), using anti-CAT (top) or anti-fLuc (bottom) Ab. Different relative amounts of cell extract were analysed to facilitate comparison of protein levels between wild-type and mutant samples.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: De novo and re-initiation of di-cistronic reporters using UAG as the initiation codon. A. Relative enzyme activity from E. coli CA274 transformed with CL or mutant di-cistronic reporters containing either a mutant CAT reporter (Cam1L) or a mutant fLuc reporter (CLam1) and expressing the U35A36 mutant initiator tRNA. Cell extracts were analysed for CAT (Cam1L, white bar) or fLuc activity (CLam1, grey bar) and compared with activity from CL. CAT activity was determined as described in Experimental procedures and normalized to total protein and β-lactamase activity (to normalize for plasmid copy number). fLuc activity was determined as described in Experimental procedures and normalized to cell number and CAT activity. B. Immunoblots of cell extracts from (A), using anti-CAT (top) or anti-fLuc (bottom) Ab. Different relative amounts of cell extract were analysed to facilitate comparison of protein levels between wild-type and mutant samples.
Mentions: Changing the CAU anticodon of wild-type initiator tRNA to CUA (U35A36 mutant) or GAC (G34C36 mutant) allows the mutant initiator tRNA to initiate from UAG and GUC codons, respectively, in E. coli (Fig. 1B) (Varshney and RajBhandary, 1990; Wu and RajBhandary, 1997). The corresponding G34C36 mutant initiator tRNA can, similarly, be used to initiate de novo protein synthesis from GUC codons in a mutant CAT gene in mammalian cells (Drabkin and RajBhandary, 1998) and in a mutant leaderless bacterio-opsin gene in the archaeon Halobacterium salinarum (Srinivasan et al., 2006). Prior to testing if UAG could also be used as a start codon for translation re-initiation, we confirmed that the mono-cistronic reporter Lam1 could be initiated from UAG by de novo initiation (∼100% efficiency, see Fig. S2). We proceeded to compare the efficiency of the U35A36 mutant initiator tRNA in de novo initiation of mutant CAT from Cam1L and re-initiation of mutant fLuc from CLam1. Based on enzyme activity, the mutant CAT reporter was translated to ∼60% efficiency, while the mutant fLuc reporter was translated to ∼7% efficiency (Fig. 4A) (the efficiencies were calculated relative to enzyme activity from the wild-type reporter, CL). We noticed, however, that there was a discrepancy between mutant CAT enzyme activity and protein levels. Immunoblot analysis showed an almost fivefold reduction in CAT protein levels in cells expressing the Cam1L reporter (Fig. 4B, top) compared with those expressing the wild-type CL reporter, instead of the ∼1.6-fold reduction expected on the basis of CAT activities (Fig. 4A). These results suggest that the specific activity of the mutant CAT protein initiated with formylglutamine and extended at the C-terminus by 13 amino acids, is higher by a factor of ∼3 than wild-type CAT protein initiated with formylmethionine, leading to an overestimation of CAT expression based on enzyme activity. To correct for this inconsistency, we used immunoblotting and densitometric analysis of the immunoblots to calculate the translation initiation efficiencies for mutant CAT expression (Table S1). Use of this assay revealed that the U35A36 mutant initiator tRNA was still more efficient in de novo initiation of mutant CAT (17%) than in re-initiation of the mutant fLuc reporter (7%). The specific activity of the fLuc reporter was not affected by the type of amino acid present at the N-terminus (compare Fig. 4A and B, bottom). Interestingly, a non-fusion, native CAT reporter previously used in our laboratory did not exhibit differences in specific activity when initiating with amino acids other than methionine (Varshney and RajBhandary, 1990; Mangroo and RajBhandary, 1995; Mayer et al., 2003). This was also verified experimentally during this work (data not shown).

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