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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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Quantitated data for various constructs in the context of the PK1+PK2_K/O mutationa, Combination of knocking out the RLUC SDS (Upstream SDS_K/O) with the PK2_K/O or PK1+PK2_K/O. Initial rates of RLUC are greatly diminished. Rates of FLUC are lower, but less diminished than RLUC. This is most likely attributable to the decreased competition for ribosomes and the presence of the SDS-like sequence upstream of the FLUC ORF and not to robust initiation on the IRES. b, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−1) mutation. c, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−2) mutation. d, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the uSTOP and uAUG mutations. Error bars are one standard deviation from the mean from three biological replicates.
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Figure 13: Quantitated data for various constructs in the context of the PK1+PK2_K/O mutationa, Combination of knocking out the RLUC SDS (Upstream SDS_K/O) with the PK2_K/O or PK1+PK2_K/O. Initial rates of RLUC are greatly diminished. Rates of FLUC are lower, but less diminished than RLUC. This is most likely attributable to the decreased competition for ribosomes and the presence of the SDS-like sequence upstream of the FLUC ORF and not to robust initiation on the IRES. b, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−1) mutation. c, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−2) mutation. d, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the uSTOP and uAUG mutations. Error bars are one standard deviation from the mean from three biological replicates.

Mentions: In eukaryotes, IGR IRES-driven translation begins directly on the IRES and is proposed to co-opt the ribosome’s elongation cycle17,19,21,22; we asked if this is true in bacteria where the IRES-ribosome interactions appear different and transient. Removal of the FLUC start codon located 15 nucleotides downstream of the IRES structure (ΔAUG) resulted in a complete loss of FLUC production, while a stop codon placed upstream of the FLUC start codon (uSTOP) had little effect (Fig. 4a&b; Extended Data Fig. 8b). Removal of 1 or 2 nucleotides just upstream of the FLUC AUG [F-SHIFT(−1) and F-SHIFT(−2)] had little effect. These results are consistent with translation in bacteria beginning on the FLUC AUG, not directly at the IRES on a non-AUG codon. This implies a repositioning of the ribosome from the IRES to the FLUC start codon. To explore this, we created a construct with an out-of-frame start codon between the IRES and the start codon (uAUG); this mutation decreased activity but not to the degree expected if this codon were being used efficiently. The source of this discrimination is not clear, but we posit that selection of the FLUC AUG is assisted by the nearly ideally positioned cryptic SDS-like sequence upstream. Constructs with alterations between the IRES and FLUC start codon all had decreased activity in the context of the PK1+PK2_K/O mutation (Extended Data Fig. 9), indicating that IRES structural integrity remains necessary for their function.


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)

Quantitated data for various constructs in the context of the PK1+PK2_K/O mutationa, Combination of knocking out the RLUC SDS (Upstream SDS_K/O) with the PK2_K/O or PK1+PK2_K/O. Initial rates of RLUC are greatly diminished. Rates of FLUC are lower, but less diminished than RLUC. This is most likely attributable to the decreased competition for ribosomes and the presence of the SDS-like sequence upstream of the FLUC ORF and not to robust initiation on the IRES. b, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−1) mutation. c, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−2) mutation. d, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the uSTOP and uAUG mutations. Error bars are one standard deviation from the mean from three biological replicates.
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getmorefigures.php?uid=PMC4352134&req=5

Figure 13: Quantitated data for various constructs in the context of the PK1+PK2_K/O mutationa, Combination of knocking out the RLUC SDS (Upstream SDS_K/O) with the PK2_K/O or PK1+PK2_K/O. Initial rates of RLUC are greatly diminished. Rates of FLUC are lower, but less diminished than RLUC. This is most likely attributable to the decreased competition for ribosomes and the presence of the SDS-like sequence upstream of the FLUC ORF and not to robust initiation on the IRES. b, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−1) mutation. c, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the FSHIFT(−2) mutation. d, The PK1+PK2_K/O dramatically reduced initial rate of FLUC production on the IRES with the uSTOP and uAUG mutations. Error bars are one standard deviation from the mean from three biological replicates.
Mentions: In eukaryotes, IGR IRES-driven translation begins directly on the IRES and is proposed to co-opt the ribosome’s elongation cycle17,19,21,22; we asked if this is true in bacteria where the IRES-ribosome interactions appear different and transient. Removal of the FLUC start codon located 15 nucleotides downstream of the IRES structure (ΔAUG) resulted in a complete loss of FLUC production, while a stop codon placed upstream of the FLUC start codon (uSTOP) had little effect (Fig. 4a&b; Extended Data Fig. 8b). Removal of 1 or 2 nucleotides just upstream of the FLUC AUG [F-SHIFT(−1) and F-SHIFT(−2)] had little effect. These results are consistent with translation in bacteria beginning on the FLUC AUG, not directly at the IRES on a non-AUG codon. This implies a repositioning of the ribosome from the IRES to the FLUC start codon. To explore this, we created a construct with an out-of-frame start codon between the IRES and the start codon (uAUG); this mutation decreased activity but not to the degree expected if this codon were being used efficiently. The source of this discrimination is not clear, but we posit that selection of the FLUC AUG is assisted by the nearly ideally positioned cryptic SDS-like sequence upstream. Constructs with alterations between the IRES and FLUC start codon all had decreased activity in the context of the PK1+PK2_K/O mutation (Extended Data Fig. 9), indicating that IRES structural integrity remains necessary for their function.

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