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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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IRES-70S ribosome structurea, Crystal structure of a full-length PSIV IGR IRES bound to T. thermophilus 70S ribosomes. Cyan: small subunit; yellow: large subunit; red: PSIV IRES domain 3; grey: density corresponding to domain 3; magenta: unbiased difference Fobs-Fcalc density corresponding to domain 1+2, with the crystal structure of PSIV IGR IRES domain 1+2 (black) docked as a rigid body26. b, Superimposition of crystal structures of the PSIV IGR IRES•70S ribosome complex (this work) and the 70S ribosome; yellow: IRES-bound 50S subunit. Domain 1+2 shifts the L1 stalk relative to its position in tRNA-bound complexes by ~15Å.
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Figure 2: IRES-70S ribosome structurea, Crystal structure of a full-length PSIV IGR IRES bound to T. thermophilus 70S ribosomes. Cyan: small subunit; yellow: large subunit; red: PSIV IRES domain 3; grey: density corresponding to domain 3; magenta: unbiased difference Fobs-Fcalc density corresponding to domain 1+2, with the crystal structure of PSIV IGR IRES domain 1+2 (black) docked as a rigid body26. b, Superimposition of crystal structures of the PSIV IGR IRES•70S ribosome complex (this work) and the 70S ribosome; yellow: IRES-bound 50S subunit. Domain 1+2 shifts the L1 stalk relative to its position in tRNA-bound complexes by ~15Å.

Mentions: To determine the structural basis for IGR IRES activity in bacteria, we solved the crystal structure of the full-length IRES RNA•70S ribosome complex to 3.8 Å resolution. In eukaryotes, IGR IRES domain 1+2 contacts both subunits, while domain 3 mimics an mRNA/tRNA interaction on the small subunit (Extended Data Fig. 1b)7,8,10,11,19,25. We observed electron density for domain 3 in the P site as in the crystal structure of isolated domain 3 bound to 70S ribosomes19 (Fig. 2a; Extended Data Fig. 7); this may represent an initiation-state or translocated IRES. Domain 1+2 density was weak but its location could be modeled using the crystal structure of unbound PSIV IGR IRES domain 1+226 (Fig. 2a). Domain 1+2’s location in the 70S ribosome differs from IGR IRES•80S ribosome complexes with domain 3 in the A site22,27. In 80S ribosomes, domain 1+2 interacts with eukaryotic-specific ribosomal protein eS25 and the L1 stalk10,11,28,29, which is structurally distinct from that in bacterial ribosomes30. In the full-length IRES•70S structure, the L1 stalk is displaced ~15Å compared to the structure containing domain 3 only. (Fig. 2b). The absence of eS25 and differences in the L1 stalk may be responsible for the partial disorder and location of the IRES. Nonetheless, the structure clearly illustrates that the compactly folded IRES can bind in the tRNA binding sites of bacterial ribosomes.


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)

IRES-70S ribosome structurea, Crystal structure of a full-length PSIV IGR IRES bound to T. thermophilus 70S ribosomes. Cyan: small subunit; yellow: large subunit; red: PSIV IRES domain 3; grey: density corresponding to domain 3; magenta: unbiased difference Fobs-Fcalc density corresponding to domain 1+2, with the crystal structure of PSIV IGR IRES domain 1+2 (black) docked as a rigid body26. b, Superimposition of crystal structures of the PSIV IGR IRES•70S ribosome complex (this work) and the 70S ribosome; yellow: IRES-bound 50S subunit. Domain 1+2 shifts the L1 stalk relative to its position in tRNA-bound complexes by ~15Å.
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Related In: Results  -  Collection

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

Figure 2: IRES-70S ribosome structurea, Crystal structure of a full-length PSIV IGR IRES bound to T. thermophilus 70S ribosomes. Cyan: small subunit; yellow: large subunit; red: PSIV IRES domain 3; grey: density corresponding to domain 3; magenta: unbiased difference Fobs-Fcalc density corresponding to domain 1+2, with the crystal structure of PSIV IGR IRES domain 1+2 (black) docked as a rigid body26. b, Superimposition of crystal structures of the PSIV IGR IRES•70S ribosome complex (this work) and the 70S ribosome; yellow: IRES-bound 50S subunit. Domain 1+2 shifts the L1 stalk relative to its position in tRNA-bound complexes by ~15Å.
Mentions: To determine the structural basis for IGR IRES activity in bacteria, we solved the crystal structure of the full-length IRES RNA•70S ribosome complex to 3.8 Å resolution. In eukaryotes, IGR IRES domain 1+2 contacts both subunits, while domain 3 mimics an mRNA/tRNA interaction on the small subunit (Extended Data Fig. 1b)7,8,10,11,19,25. We observed electron density for domain 3 in the P site as in the crystal structure of isolated domain 3 bound to 70S ribosomes19 (Fig. 2a; Extended Data Fig. 7); this may represent an initiation-state or translocated IRES. Domain 1+2 density was weak but its location could be modeled using the crystal structure of unbound PSIV IGR IRES domain 1+226 (Fig. 2a). Domain 1+2’s location in the 70S ribosome differs from IGR IRES•80S ribosome complexes with domain 3 in the A site22,27. In 80S ribosomes, domain 1+2 interacts with eukaryotic-specific ribosomal protein eS25 and the L1 stalk10,11,28,29, which is structurally distinct from that in bacterial ribosomes30. In the full-length IRES•70S structure, the L1 stalk is displaced ~15Å compared to the structure containing domain 3 only. (Fig. 2b). The absence of eS25 and differences in the L1 stalk may be responsible for the partial disorder and location of the IRES. Nonetheless, the structure clearly illustrates that the compactly folded IRES can bind in the tRNA binding sites of bacterial ribosomes.

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
Related in: MedlinePlus