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Regulation of translation in haloarchaea: 5'- and 3'-UTRs are essential and have to functionally interact in vivo.

Brenneis M, Soppa J - PLoS ONE (2009)

Bottom Line: Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis.An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR.The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Goethe-University, Institute for Molecular Biosciences, Frankfurt, Germany.

ABSTRACT
Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

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Two differentially translated H. volcanii genes and their UTRs.(A) The lengths of the 5′- and 3′-UTRs of the hp and the hlr transcripts are tabulated. The UTRs of the genes were determined in a prior study [22]. (B) The sequences of the 5′- and 3′-UTRs of the hlr and the hp transcripts are shown. The start as well as the stop codon of the orf are also included and printed in bold. (C) Growth phase-dependent differential translational efficiencies of the hlr and hp genes. The values were obtained by isolating free, non-translated RNAs as well as polysome-bound RNAs and their genome-wide comparison with DNA microarrays [7]. The results were normalized to the average of all genes. The ratio of polysomal to free RNA for the hlr and hp transcripts are shown for exponentially growing and stationary phase cells.
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pone-0004484-g001: Two differentially translated H. volcanii genes and their UTRs.(A) The lengths of the 5′- and 3′-UTRs of the hp and the hlr transcripts are tabulated. The UTRs of the genes were determined in a prior study [22]. (B) The sequences of the 5′- and 3′-UTRs of the hlr and the hp transcripts are shown. The start as well as the stop codon of the orf are also included and printed in bold. (C) Growth phase-dependent differential translational efficiencies of the hlr and hp genes. The values were obtained by isolating free, non-translated RNAs as well as polysome-bound RNAs and their genome-wide comparison with DNA microarrays [7]. The results were normalized to the average of all genes. The ratio of polysomal to free RNA for the hlr and hp transcripts are shown for exponentially growing and stationary phase cells.

Mentions: Two genes were chosen to characterize the in vivo roles of 5′- and 3′-UTRs in H. volcanii, i.e. the gene HVO_2837 (www.halolex.mpg.de; archaea.ucsc.edu) encoding a “hoxA like transcriptional regulator” (hlr) and the gene HVO_0721 encoding a “conserved hypothetical protein” (hp). It was shown previously that the native transcripts of both genes contain a 5′-UTR lacking a Shine-Dalgarno (SD) sequence and a 3′-UTR of average length [22]. The lengths and sequences of the UTRs are summarized in Figure 1A and B. Global analyses had revealed that the transcripts of both genes exhibit differential growth phase-dependent translational efficiencies [7]. The translational efficiency of the hlr transcript is down-regulated in exponential growth phase, while, in contrast, the translational efficiency of the hp transcript is down-regulated in stationary growth phase (Figure 1C). Previously translational regulation was determined by quantifying the fractions of free and polysome-bound transcripts using DNA microarrays, which is time-consuming and confined to native transcripts. Therefore, in the current study a reporter gene system was used to determine translational efficiencies. Transcript levels were quantified by RT-Real Time PCR and protein levels were quantified using an enzymatic test. The 5′- and 3′-UTRs of the two transcripts were fused to the dhfr reporter gene, either alone or simultaneously. As a control, the leaderless dhfr was used without its native 3′-UTR. The different transcript variants are schematically outlined in Figure 2A (all plasmids used in this study are summarized in Table 1). H. volcanii cultures transformed with the respective plasmids were grown to exponential growth phase (2×108 cells/ml) and to stationary phase (2×109 cells/ml). The dhfr transcript levels as well as the DHFR specific activities were determined and the translational efficiencies were calculated (Figure 2A). The results were normalized to the control transcript and are visualized in Figure 2B.


Regulation of translation in haloarchaea: 5'- and 3'-UTRs are essential and have to functionally interact in vivo.

Brenneis M, Soppa J - PLoS ONE (2009)

Two differentially translated H. volcanii genes and their UTRs.(A) The lengths of the 5′- and 3′-UTRs of the hp and the hlr transcripts are tabulated. The UTRs of the genes were determined in a prior study [22]. (B) The sequences of the 5′- and 3′-UTRs of the hlr and the hp transcripts are shown. The start as well as the stop codon of the orf are also included and printed in bold. (C) Growth phase-dependent differential translational efficiencies of the hlr and hp genes. The values were obtained by isolating free, non-translated RNAs as well as polysome-bound RNAs and their genome-wide comparison with DNA microarrays [7]. The results were normalized to the average of all genes. The ratio of polysomal to free RNA for the hlr and hp transcripts are shown for exponentially growing and stationary phase cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636863&req=5

pone-0004484-g001: Two differentially translated H. volcanii genes and their UTRs.(A) The lengths of the 5′- and 3′-UTRs of the hp and the hlr transcripts are tabulated. The UTRs of the genes were determined in a prior study [22]. (B) The sequences of the 5′- and 3′-UTRs of the hlr and the hp transcripts are shown. The start as well as the stop codon of the orf are also included and printed in bold. (C) Growth phase-dependent differential translational efficiencies of the hlr and hp genes. The values were obtained by isolating free, non-translated RNAs as well as polysome-bound RNAs and their genome-wide comparison with DNA microarrays [7]. The results were normalized to the average of all genes. The ratio of polysomal to free RNA for the hlr and hp transcripts are shown for exponentially growing and stationary phase cells.
Mentions: Two genes were chosen to characterize the in vivo roles of 5′- and 3′-UTRs in H. volcanii, i.e. the gene HVO_2837 (www.halolex.mpg.de; archaea.ucsc.edu) encoding a “hoxA like transcriptional regulator” (hlr) and the gene HVO_0721 encoding a “conserved hypothetical protein” (hp). It was shown previously that the native transcripts of both genes contain a 5′-UTR lacking a Shine-Dalgarno (SD) sequence and a 3′-UTR of average length [22]. The lengths and sequences of the UTRs are summarized in Figure 1A and B. Global analyses had revealed that the transcripts of both genes exhibit differential growth phase-dependent translational efficiencies [7]. The translational efficiency of the hlr transcript is down-regulated in exponential growth phase, while, in contrast, the translational efficiency of the hp transcript is down-regulated in stationary growth phase (Figure 1C). Previously translational regulation was determined by quantifying the fractions of free and polysome-bound transcripts using DNA microarrays, which is time-consuming and confined to native transcripts. Therefore, in the current study a reporter gene system was used to determine translational efficiencies. Transcript levels were quantified by RT-Real Time PCR and protein levels were quantified using an enzymatic test. The 5′- and 3′-UTRs of the two transcripts were fused to the dhfr reporter gene, either alone or simultaneously. As a control, the leaderless dhfr was used without its native 3′-UTR. The different transcript variants are schematically outlined in Figure 2A (all plasmids used in this study are summarized in Table 1). H. volcanii cultures transformed with the respective plasmids were grown to exponential growth phase (2×108 cells/ml) and to stationary phase (2×109 cells/ml). The dhfr transcript levels as well as the DHFR specific activities were determined and the translational efficiencies were calculated (Figure 2A). The results were normalized to the control transcript and are visualized in Figure 2B.

Bottom Line: Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis.An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR.The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Goethe-University, Institute for Molecular Biosciences, Frankfurt, Germany.

ABSTRACT
Recently a first genome-wide analysis of translational regulation using prokaryotic species had been performed which revealed that regulation of translational efficiency plays an important role in haloarchaea. In fact, the fractions of genes under differential growth phase-dependent translational control in the two species Halobacterium salinarum and Haloferax volcanii were as high as in eukaryotes. However, nothing is known about the mechanisms of translational regulation in archaea. Therefore, two genes exhibiting opposing directions of regulation were selected to unravel the importance of untranslated regions (UTRs) for differential translational control in vivo.Differential translational regulation in exponentially growing versus stationary phase cells was studied by comparing translational efficiencies using a reporter gene system. Translational regulation was not observed when 5'-UTRs or 3'-UTRs alone were fused to the reporter gene. However, their simultaneous presence was sufficient to transfer differential translational control from the native transcript to the reporter transcript. This was true for both directions of translational control. Translational regulation was completely abolished when stem loops in the 5'-UTR were changed by mutagenesis. An "UTR-swap" experiment demonstrated that the direction of translational regulation is encoded in the 3'-UTR, not in the 5'-UTR. While much is known about 5'-UTR-dependent translational control in bacteria, the reported findings provide the first examples that both 5'- and 3'-UTRs are essential and sufficient to drive differential translational regulation in a prokaryote and therefore have to functionally interact in vivo. The current results indicate that 3'-UTR-dependent translational control had already evolved before capping and polyadenylation of transcripts were invented, which are essential for circularization of transcripts in eukaryotes.

Show MeSH