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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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Contributions of region upstream of AUG to initiation activitya, Diagram of constructs tested and traces of FLUC and RLUC production. The y axis shows relative light units (RLUs). b, Quantitated initial rates from these constructs. Results from CSFV IRES (negative control) shown for comparison. “Downstream SDS” contains an SDS driving FLUC production (in place of the IRES), “Downstream SDS-like” contains the purine-rich sequence in place of the IRES and driving FLUC production. In “Downstream SDS-like_K/O”, this purine-rich sequence has been replaced by a pyrimidine-rich sequence. A PSIV IRES construct in which both pseudoknots are disrupted and the purine-rich SDS-like sequence just downstream of the IRES is mutated has essentially the same activity as the CSFV IRES (Downstream SDS-like_K/O+PK1+PK2_K/O). Error bars are one standard deviation from the mean of three biological replicates.
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Figure 10: Contributions of region upstream of AUG to initiation activitya, Diagram of constructs tested and traces of FLUC and RLUC production. The y axis shows relative light units (RLUs). b, Quantitated initial rates from these constructs. Results from CSFV IRES (negative control) shown for comparison. “Downstream SDS” contains an SDS driving FLUC production (in place of the IRES), “Downstream SDS-like” contains the purine-rich sequence in place of the IRES and driving FLUC production. In “Downstream SDS-like_K/O”, this purine-rich sequence has been replaced by a pyrimidine-rich sequence. A PSIV IRES construct in which both pseudoknots are disrupted and the purine-rich SDS-like sequence just downstream of the IRES is mutated has essentially the same activity as the CSFV IRES (Downstream SDS-like_K/O+PK1+PK2_K/O). Error bars are one standard deviation from the mean of three biological replicates.

Mentions: A source of initiation from the IGR IRES could be a “cryptic” SDS in the purine-rich sequence between the IRES and the FLUC start codon (Extended Data Fig. 6). FLUC production from this SDS-like sequence alone was at ~30% of the WT IRES, not enough to account for all FLUC produced from the IRES. Mutating this SDS-like sequence in the context of the full IRES decreased FLUC production, but translation was still higher than from an SDS or the SDS-like sequence. Thus, the structured IRES can drive FLUC production without the SDS-like sequence, but both likely contribute to function when present.


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)

Contributions of region upstream of AUG to initiation activitya, Diagram of constructs tested and traces of FLUC and RLUC production. The y axis shows relative light units (RLUs). b, Quantitated initial rates from these constructs. Results from CSFV IRES (negative control) shown for comparison. “Downstream SDS” contains an SDS driving FLUC production (in place of the IRES), “Downstream SDS-like” contains the purine-rich sequence in place of the IRES and driving FLUC production. In “Downstream SDS-like_K/O”, this purine-rich sequence has been replaced by a pyrimidine-rich sequence. A PSIV IRES construct in which both pseudoknots are disrupted and the purine-rich SDS-like sequence just downstream of the IRES is mutated has essentially the same activity as the CSFV IRES (Downstream SDS-like_K/O+PK1+PK2_K/O). Error bars are one standard deviation from the mean of three biological replicates.
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Related In: Results  -  Collection

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

Figure 10: Contributions of region upstream of AUG to initiation activitya, Diagram of constructs tested and traces of FLUC and RLUC production. The y axis shows relative light units (RLUs). b, Quantitated initial rates from these constructs. Results from CSFV IRES (negative control) shown for comparison. “Downstream SDS” contains an SDS driving FLUC production (in place of the IRES), “Downstream SDS-like” contains the purine-rich sequence in place of the IRES and driving FLUC production. In “Downstream SDS-like_K/O”, this purine-rich sequence has been replaced by a pyrimidine-rich sequence. A PSIV IRES construct in which both pseudoknots are disrupted and the purine-rich SDS-like sequence just downstream of the IRES is mutated has essentially the same activity as the CSFV IRES (Downstream SDS-like_K/O+PK1+PK2_K/O). Error bars are one standard deviation from the mean of three biological replicates.
Mentions: A source of initiation from the IGR IRES could be a “cryptic” SDS in the purine-rich sequence between the IRES and the FLUC start codon (Extended Data Fig. 6). FLUC production from this SDS-like sequence alone was at ~30% of the WT IRES, not enough to account for all FLUC produced from the IRES. Mutating this SDS-like sequence in the context of the full IRES decreased FLUC production, but translation was still higher than from an SDS or the SDS-like sequence. Thus, the structured IRES can drive FLUC production without the SDS-like sequence, but both likely contribute to function when present.

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