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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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PSIV IGR IRES sequence, secondary structure, and design of mutantsa, Secondary structure of the full-length IGR IRES from the Plautia stali intestine virus (PSIV). The specific changes that were introduced to generate the mutants and constructs described and tested in the main text are shown. For each, the altered region is boxed and the change is shown in red. For the uAUG and uSTOP constructs, the start and stop codons are underlined. RLUC and FLUC coding sequences are boxed cyan and yellow, respectively. b, Constructs without the IRES that contain various wild-type or mutant SDS and SDS-like sequences upstream of the FLUC ORF. c, Construct containing just domain 3 of the PSIV IGR IRES.
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Figure 9: PSIV IGR IRES sequence, secondary structure, and design of mutantsa, Secondary structure of the full-length IGR IRES from the Plautia stali intestine virus (PSIV). The specific changes that were introduced to generate the mutants and constructs described and tested in the main text are shown. For each, the altered region is boxed and the change is shown in red. For the uAUG and uSTOP constructs, the start and stop codons are underlined. RLUC and FLUC coding sequences are boxed cyan and yellow, respectively. b, Constructs without the IRES that contain various wild-type or mutant SDS and SDS-like sequences upstream of the FLUC ORF. c, Construct containing just domain 3 of the PSIV IGR IRES.

Mentions: We generated an inducible expression vector encoding a single mRNA containing two independent luciferase (LUC) reporters (Extended Data Fig. 1d)24, and verified that it allowed simultaneous measurement of initial rates of production of each protein (Extended Data Fig. 2&3). We used this construct to test if an IGR IRES RNA can drive translation in live bacteria. The Renilla luciferase (RLUC) was placed to initiate translation from a SDS (and “enhancer” sequence), and the Firefly luciferase (FLUC) was placed after a Wild-type (WT) Plautia stali intestine virus (PSIV) IGR IRES. There was some production of both LUCs prior to induction (due to expected “leaky expression”, Extended Data Fig. 4), but induction resulted in marked increase in both reporters; the production of FLUC is consistent with translation beginning at the IRES (Fig. 1c; Extended Data Fig. 2). Removing the RLUC-driving SDS (Upstream SDS_K/O; all mutants shown in Extended Data Fig. 5) diminished production of RLUC, but FLUC production increased >10-fold (Fig. 1b; all raw LUC data in Extended Data Table 1a), attributable to decreased competition for ribosomes and with ribosomes initiating independently at the IRES. Replacing the IGR IRES with the IRES from classical swine fever virus (CSFV) resulted in negligible FLUC production (Extended Data Fig. 2), demonstrating specificity for the IGR IRES.


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)

PSIV IGR IRES sequence, secondary structure, and design of mutantsa, Secondary structure of the full-length IGR IRES from the Plautia stali intestine virus (PSIV). The specific changes that were introduced to generate the mutants and constructs described and tested in the main text are shown. For each, the altered region is boxed and the change is shown in red. For the uAUG and uSTOP constructs, the start and stop codons are underlined. RLUC and FLUC coding sequences are boxed cyan and yellow, respectively. b, Constructs without the IRES that contain various wild-type or mutant SDS and SDS-like sequences upstream of the FLUC ORF. c, Construct containing just domain 3 of the PSIV IGR IRES.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: PSIV IGR IRES sequence, secondary structure, and design of mutantsa, Secondary structure of the full-length IGR IRES from the Plautia stali intestine virus (PSIV). The specific changes that were introduced to generate the mutants and constructs described and tested in the main text are shown. For each, the altered region is boxed and the change is shown in red. For the uAUG and uSTOP constructs, the start and stop codons are underlined. RLUC and FLUC coding sequences are boxed cyan and yellow, respectively. b, Constructs without the IRES that contain various wild-type or mutant SDS and SDS-like sequences upstream of the FLUC ORF. c, Construct containing just domain 3 of the PSIV IGR IRES.
Mentions: We generated an inducible expression vector encoding a single mRNA containing two independent luciferase (LUC) reporters (Extended Data Fig. 1d)24, and verified that it allowed simultaneous measurement of initial rates of production of each protein (Extended Data Fig. 2&3). We used this construct to test if an IGR IRES RNA can drive translation in live bacteria. The Renilla luciferase (RLUC) was placed to initiate translation from a SDS (and “enhancer” sequence), and the Firefly luciferase (FLUC) was placed after a Wild-type (WT) Plautia stali intestine virus (PSIV) IGR IRES. There was some production of both LUCs prior to induction (due to expected “leaky expression”, Extended Data Fig. 4), but induction resulted in marked increase in both reporters; the production of FLUC is consistent with translation beginning at the IRES (Fig. 1c; Extended Data Fig. 2). Removing the RLUC-driving SDS (Upstream SDS_K/O; all mutants shown in Extended Data Fig. 5) diminished production of RLUC, but FLUC production increased >10-fold (Fig. 1b; all raw LUC data in Extended Data Table 1a), attributable to decreased competition for ribosomes and with ribosomes initiating independently at the IRES. Replacing the IGR IRES with the IRES from classical swine fever virus (CSFV) resulted in negligible FLUC production (Extended Data Fig. 2), demonstrating specificity for the IGR IRES.

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