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Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

Colussi TM, Costantino DA, Zhu J, Donohue JP, Korostelev AA, Jaafar ZA, Plank TD, Noller HF, Kieft JS - Nature (2015)

Bottom Line: However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life.We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs.This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA [2] Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.

ABSTRACT
The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

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Luciferase activity time-courses for various constructsa, Time-course traces for constructs and bar graphs shown in Fig. 3. b, Time-course traces for constructs and bar graphs shown in Fig. 4. Error bars are one standard deviation from the mean of three biological replicates. In both panels, the y axis shows relative light units (RLUs).
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Figure 12: Luciferase activity time-courses for various constructsa, Time-course traces for constructs and bar graphs shown in Fig. 3. b, Time-course traces for constructs and bar graphs shown in Fig. 4. Error bars are one standard deviation from the mean of three biological replicates. In both panels, the y axis shows relative light units (RLUs).

Mentions: The IGR IRES’ compact structure is essential for function in eukaryotes25,26, and the IRES•70S structure suggested this is true in bacteria. To test this, we disrupted two pseudoknots essential for the IRES’ compact structure, both individually (PK1_K/O, PK2_K/O) and together (PK1+PK2_K/O) and measured activity (Fig. 3a&b; Extended Data Fig. 8a)10. FLUC production decreased in all three, with the double mutant at a level where activity could be accounted for by the cryptic SDS-like sequence. Indeed, disruption of both pseudoknots and the SDS-like sequence (Downstream SDS-like_K/O+PK1+PK2_K/O) abrogated IRES activity (Extended Data Fig. 6). Isolated IRES domain 3 operated similarly to the domain 1+2-disrupting mutant (PK2_K/O). Thus, IGR IRES translation in bacteria depends on a compact RNA structure and although domain 1+2 is poorly ordered in the crystal, it may be required to form transient interactions with the ribosome.


Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

Colussi TM, Costantino DA, Zhu J, Donohue JP, Korostelev AA, Jaafar ZA, Plank TD, Noller HF, Kieft JS - Nature (2015)

Luciferase activity time-courses for various constructsa, Time-course traces for constructs and bar graphs shown in Fig. 3. b, Time-course traces for constructs and bar graphs shown in Fig. 4. Error bars are one standard deviation from the mean of three biological replicates. In both panels, the y axis shows relative light units (RLUs).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352134&req=5

Figure 12: Luciferase activity time-courses for various constructsa, Time-course traces for constructs and bar graphs shown in Fig. 3. b, Time-course traces for constructs and bar graphs shown in Fig. 4. Error bars are one standard deviation from the mean of three biological replicates. In both panels, the y axis shows relative light units (RLUs).
Mentions: The IGR IRES’ compact structure is essential for function in eukaryotes25,26, and the IRES•70S structure suggested this is true in bacteria. To test this, we disrupted two pseudoknots essential for the IRES’ compact structure, both individually (PK1_K/O, PK2_K/O) and together (PK1+PK2_K/O) and measured activity (Fig. 3a&b; Extended Data Fig. 8a)10. FLUC production decreased in all three, with the double mutant at a level where activity could be accounted for by the cryptic SDS-like sequence. Indeed, disruption of both pseudoknots and the SDS-like sequence (Downstream SDS-like_K/O+PK1+PK2_K/O) abrogated IRES activity (Extended Data Fig. 6). Isolated IRES domain 3 operated similarly to the domain 1+2-disrupting mutant (PK2_K/O). Thus, IGR IRES translation in bacteria depends on a compact RNA structure and although domain 1+2 is poorly ordered in the crystal, it may be required to form transient interactions with the ribosome.

Bottom Line: However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life.We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs.This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA [2] Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.

ABSTRACT
The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Show MeSH