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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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SSU intermediates purified with all three tags contain 17S rRNA as a major pre-16S rRNA species(a) RNA analysis of rRNA purified with tags 105L, 11L and 20T on a 2% denaturing agarose gel stained with ethidium bromide. The three elution fractions with highest amounts of RNA from representative affinity purification are shown. Uncropped images are shown in Supplementary Data Set 3. (b) Northern blot analysis using probes directed to leader, trailer and mature regions of 16S rRNA for the same fractions as in Panel a. Positions of the probe are indicated below. Uncropped images are shown in Supplementary Data Set 3. (c) RT-PCR (Reverse Transcription – Polymerase Chain Reaction) products of the ligated junctions of pre-16S rRNA purified with the three tags (modified 3′5′RACE; see Methods) resolved on a 2% agarose gel and stained with ethidium bromide. Only one fraction from the above analysis is shown; similar results are obtained for all elution fractions. Ŧ indicates heat treatment of RNA before ligation. The experiments in panel a, b and c were carried out at least four times.(d) Modified 3′5′ RACE products of pre-16S RNA purified with tag 11L from the Δrng strain of E. coli, which has been previously shown to accumulate 16.3S rRNA species, processed by RNase E at the 5′ end13. 16.3S rRNA is marked. Uncropped image is shown in Supplementary Data Set 3. The identity of the products is confirmed by sequencing (data not shown). The experiments were carried out two times.
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Figure 2: SSU intermediates purified with all three tags contain 17S rRNA as a major pre-16S rRNA species(a) RNA analysis of rRNA purified with tags 105L, 11L and 20T on a 2% denaturing agarose gel stained with ethidium bromide. The three elution fractions with highest amounts of RNA from representative affinity purification are shown. Uncropped images are shown in Supplementary Data Set 3. (b) Northern blot analysis using probes directed to leader, trailer and mature regions of 16S rRNA for the same fractions as in Panel a. Positions of the probe are indicated below. Uncropped images are shown in Supplementary Data Set 3. (c) RT-PCR (Reverse Transcription – Polymerase Chain Reaction) products of the ligated junctions of pre-16S rRNA purified with the three tags (modified 3′5′RACE; see Methods) resolved on a 2% agarose gel and stained with ethidium bromide. Only one fraction from the above analysis is shown; similar results are obtained for all elution fractions. Ŧ indicates heat treatment of RNA before ligation. The experiments in panel a, b and c were carried out at least four times.(d) Modified 3′5′ RACE products of pre-16S RNA purified with tag 11L from the Δrng strain of E. coli, which has been previously shown to accumulate 16.3S rRNA species, processed by RNase E at the 5′ end13. 16.3S rRNA is marked. Uncropped image is shown in Supplementary Data Set 3. The identity of the products is confirmed by sequencing (data not shown). The experiments were carried out two times.

Mentions: Examination of pre-rRNA from the purified RNPs by denaturing gel electrophoresis (Fig. 2a), Northern blotting (Fig. 2b), modified 3′5′ RACE (Rapid amplification of cDNA ends) (Fig. 2c) and primer extension (data not shown) revealed that the predominant purified rRNA was 17S rRNA, the pre-SSU species liberated from the primary rRNA transcript by RNase III cleavage. Purification of assembly intermediates tagged at position 11L from an E. coli strain lacking RNase G, a 16S rRNA maturation enzyme, resulted in isolation of significant amounts of 16.3S rRNA (compared to the 17S rRNA) (Fig. 2d), consistent with previous results on pre-rRNA processing in this strain12,22. These data indicate that affinity purification can yield many pre-16S rRNA containing rRNPs and thus suggests that isolation of 17S rRNA from several tagged positions in wild-type E. coli is not due to other species being refractory to purification and suggest that 17S rRNA is a platform for assembly of pre-SSUs in the wild-type E. coli.


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)

SSU intermediates purified with all three tags contain 17S rRNA as a major pre-16S rRNA species(a) RNA analysis of rRNA purified with tags 105L, 11L and 20T on a 2% denaturing agarose gel stained with ethidium bromide. The three elution fractions with highest amounts of RNA from representative affinity purification are shown. Uncropped images are shown in Supplementary Data Set 3. (b) Northern blot analysis using probes directed to leader, trailer and mature regions of 16S rRNA for the same fractions as in Panel a. Positions of the probe are indicated below. Uncropped images are shown in Supplementary Data Set 3. (c) RT-PCR (Reverse Transcription – Polymerase Chain Reaction) products of the ligated junctions of pre-16S rRNA purified with the three tags (modified 3′5′RACE; see Methods) resolved on a 2% agarose gel and stained with ethidium bromide. Only one fraction from the above analysis is shown; similar results are obtained for all elution fractions. Ŧ indicates heat treatment of RNA before ligation. The experiments in panel a, b and c were carried out at least four times.(d) Modified 3′5′ RACE products of pre-16S RNA purified with tag 11L from the Δrng strain of E. coli, which has been previously shown to accumulate 16.3S rRNA species, processed by RNase E at the 5′ end13. 16.3S rRNA is marked. Uncropped image is shown in Supplementary Data Set 3. The identity of the products is confirmed by sequencing (data not shown). The experiments were carried out two times.
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Related In: Results  -  Collection

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Figure 2: SSU intermediates purified with all three tags contain 17S rRNA as a major pre-16S rRNA species(a) RNA analysis of rRNA purified with tags 105L, 11L and 20T on a 2% denaturing agarose gel stained with ethidium bromide. The three elution fractions with highest amounts of RNA from representative affinity purification are shown. Uncropped images are shown in Supplementary Data Set 3. (b) Northern blot analysis using probes directed to leader, trailer and mature regions of 16S rRNA for the same fractions as in Panel a. Positions of the probe are indicated below. Uncropped images are shown in Supplementary Data Set 3. (c) RT-PCR (Reverse Transcription – Polymerase Chain Reaction) products of the ligated junctions of pre-16S rRNA purified with the three tags (modified 3′5′RACE; see Methods) resolved on a 2% agarose gel and stained with ethidium bromide. Only one fraction from the above analysis is shown; similar results are obtained for all elution fractions. Ŧ indicates heat treatment of RNA before ligation. The experiments in panel a, b and c were carried out at least four times.(d) Modified 3′5′ RACE products of pre-16S RNA purified with tag 11L from the Δrng strain of E. coli, which has been previously shown to accumulate 16.3S rRNA species, processed by RNase E at the 5′ end13. 16.3S rRNA is marked. Uncropped image is shown in Supplementary Data Set 3. The identity of the products is confirmed by sequencing (data not shown). The experiments were carried out two times.
Mentions: Examination of pre-rRNA from the purified RNPs by denaturing gel electrophoresis (Fig. 2a), Northern blotting (Fig. 2b), modified 3′5′ RACE (Rapid amplification of cDNA ends) (Fig. 2c) and primer extension (data not shown) revealed that the predominant purified rRNA was 17S rRNA, the pre-SSU species liberated from the primary rRNA transcript by RNase III cleavage. Purification of assembly intermediates tagged at position 11L from an E. coli strain lacking RNase G, a 16S rRNA maturation enzyme, resulted in isolation of significant amounts of 16.3S rRNA (compared to the 17S rRNA) (Fig. 2d), consistent with previous results on pre-rRNA processing in this strain12,22. These data indicate that affinity purification can yield many pre-16S rRNA containing rRNPs and thus suggests that isolation of 17S rRNA from several tagged positions in wild-type E. coli is not due to other species being refractory to purification and suggest that 17S rRNA is a platform for assembly of pre-SSUs in the wild-type E. coli.

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
Related in: MedlinePlus