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Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA.

Gupta N, Culver GM - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle.This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli.These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, New York, USA.

ABSTRACT
Processing of transcribed precursor ribosomal RNA (pre-rRNA) to a mature state is a conserved aspect of ribosome biogenesis in vivo. We developed an affinity-purification system to isolate and analyze in vivo-formed pre-rRNA-containing ribonucleoprotein (RNP) particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre-small subunit (pre-SSU) rRNA is a platform for biogenesis. These pre-SSU-containing RNPs have differing ribosomal-protein and auxiliary factor association and rRNA folding. Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle. This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli. These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

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Maturation of 17S rRNA can initiate either at the 5′ or 3′ ends in vivo and in vitro(a) Modified 3′5′ RACE products from the three purified intermediates separated on a 2% agarose gel. Intermediate pre-16S rRNA species are indicated by an arrow and is observed in all purifications (n > 10). 17S rRNA and intermediate pre-16S rRNA are gel purified and PCR amplified and products are resolved on a 2% agarose gel marked as Product 1 and Product 2. (b) Sequencing results of the various pre-16S rRNA processing species associated with the three different purified SSU intermediates products, each product is given a designation (a–d). The experiments were carried out two times. (c) Modified 3′5′ RACE products that result from incubation of the purified 105L, 11L and 20T assembly intermediates with wild-type extracts for 0 and 60 minutes (in vitro). ‘+’ lanes indicate addition of S100 and incubation time (in mins) is shown. The experiments were carried out three times. Uncropped image is shown in Supplementary Data Set 3. Various pre-16S rRNA species are marked as in Panel b.
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Figure 5: Maturation of 17S rRNA can initiate either at the 5′ or 3′ ends in vivo and in vitro(a) Modified 3′5′ RACE products from the three purified intermediates separated on a 2% agarose gel. Intermediate pre-16S rRNA species are indicated by an arrow and is observed in all purifications (n > 10). 17S rRNA and intermediate pre-16S rRNA are gel purified and PCR amplified and products are resolved on a 2% agarose gel marked as Product 1 and Product 2. (b) Sequencing results of the various pre-16S rRNA processing species associated with the three different purified SSU intermediates products, each product is given a designation (a–d). The experiments were carried out two times. (c) Modified 3′5′ RACE products that result from incubation of the purified 105L, 11L and 20T assembly intermediates with wild-type extracts for 0 and 60 minutes (in vitro). ‘+’ lanes indicate addition of S100 and incubation time (in mins) is shown. The experiments were carried out three times. Uncropped image is shown in Supplementary Data Set 3. Various pre-16S rRNA species are marked as in Panel b.

Mentions: Our results, and data from other groups, suggest multiple pathways for r-protein association and rRNA folding during SSU assembly in vivo43,44. However, whether further maturation of 17S rRNA to 16S rRNA also follows multiple or an obligatory pathway was not known. Modified 3′5′ RACE revealed a predominant 17S rRNA species purified using all three tags; however, minor, but detectable products of lengths between 17S rRNA and 16S rRNA were also observed (Fig. 2c). These minor products represented additional pre-16S rRNA maturation products (Fig. 5a, b), with either a mature 3′ end (in 105L and 11L tagged intermediates) or a mature 5′ end (in tag 20T intermediates). These data suggest that further maturation of 17S rRNA can be initiated at either the 5′ or 3′ end of 17S rRNA in wild-type E. coli. These results show that post-RNase III cleavage, 17S rRNA processing in vivo can initiate at either end and like r-protein addition and rRNA folding, rRNA maturation can follow multiple pathways.


Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA.

Gupta N, Culver GM - Nat. Struct. Mol. Biol. (2014)

Maturation of 17S rRNA can initiate either at the 5′ or 3′ ends in vivo and in vitro(a) Modified 3′5′ RACE products from the three purified intermediates separated on a 2% agarose gel. Intermediate pre-16S rRNA species are indicated by an arrow and is observed in all purifications (n > 10). 17S rRNA and intermediate pre-16S rRNA are gel purified and PCR amplified and products are resolved on a 2% agarose gel marked as Product 1 and Product 2. (b) Sequencing results of the various pre-16S rRNA processing species associated with the three different purified SSU intermediates products, each product is given a designation (a–d). The experiments were carried out two times. (c) Modified 3′5′ RACE products that result from incubation of the purified 105L, 11L and 20T assembly intermediates with wild-type extracts for 0 and 60 minutes (in vitro). ‘+’ lanes indicate addition of S100 and incubation time (in mins) is shown. The experiments were carried out three times. Uncropped image is shown in Supplementary Data Set 3. Various pre-16S rRNA species are marked as in Panel b.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4355579&req=5

Figure 5: Maturation of 17S rRNA can initiate either at the 5′ or 3′ ends in vivo and in vitro(a) Modified 3′5′ RACE products from the three purified intermediates separated on a 2% agarose gel. Intermediate pre-16S rRNA species are indicated by an arrow and is observed in all purifications (n > 10). 17S rRNA and intermediate pre-16S rRNA are gel purified and PCR amplified and products are resolved on a 2% agarose gel marked as Product 1 and Product 2. (b) Sequencing results of the various pre-16S rRNA processing species associated with the three different purified SSU intermediates products, each product is given a designation (a–d). The experiments were carried out two times. (c) Modified 3′5′ RACE products that result from incubation of the purified 105L, 11L and 20T assembly intermediates with wild-type extracts for 0 and 60 minutes (in vitro). ‘+’ lanes indicate addition of S100 and incubation time (in mins) is shown. The experiments were carried out three times. Uncropped image is shown in Supplementary Data Set 3. Various pre-16S rRNA species are marked as in Panel b.
Mentions: Our results, and data from other groups, suggest multiple pathways for r-protein association and rRNA folding during SSU assembly in vivo43,44. However, whether further maturation of 17S rRNA to 16S rRNA also follows multiple or an obligatory pathway was not known. Modified 3′5′ RACE revealed a predominant 17S rRNA species purified using all three tags; however, minor, but detectable products of lengths between 17S rRNA and 16S rRNA were also observed (Fig. 2c). These minor products represented additional pre-16S rRNA maturation products (Fig. 5a, b), with either a mature 3′ end (in 105L and 11L tagged intermediates) or a mature 5′ end (in tag 20T intermediates). These data suggest that further maturation of 17S rRNA can be initiated at either the 5′ or 3′ end of 17S rRNA in wild-type E. coli. These results show that post-RNase III cleavage, 17S rRNA processing in vivo can initiate at either end and like r-protein addition and rRNA folding, rRNA maturation can follow multiple pathways.

Bottom Line: Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle.This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli.These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, New York, USA.

ABSTRACT
Processing of transcribed precursor ribosomal RNA (pre-rRNA) to a mature state is a conserved aspect of ribosome biogenesis in vivo. We developed an affinity-purification system to isolate and analyze in vivo-formed pre-rRNA-containing ribonucleoprotein (RNP) particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre-small subunit (pre-SSU) rRNA is a platform for biogenesis. These pre-SSU-containing RNPs have differing ribosomal-protein and auxiliary factor association and rRNA folding. Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle. This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli. These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

Show MeSH