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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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Multiple pathways for ribosomal proteins addition to the three intermediates(a) Spectral counts of r-proteins bound to intermediates are compared to mature ribosomes purified with tag at position 86M (see Methods, Supplementary Data Set 2) and relative r-protein levels are calculated as percentages [biological replicates (n = 3) and technical replicates (n = 2)]. R-protein percentages of each intermediate are binned into 4 groups and plotted as heat maps on the three-dimensional crystal structure of SSU (PDB: 2AVY)34. Solvent side is shown (see Supplementary Fig. 4a for interface side). (b) Hierarchical clustering analysis of r-proteins bound to the 105L, 11L, and 20T tagged intermediates. Relative protein abundance (RPA) for each r-protein present in each of the three SSU intermediates (but not mature SSUs) is calculated as described in Methods. Different shades of green indicate the levels of occupancy by a specific r-protein among the intermediates. The darker the green color, the more abundant the r-protein is bound in that intermediate as compared to the other intermediates. The r-proteins are clustered into three major groups, marked as I, II and III. (c) RPA of r-proteins associated with three intermediates is plotted on the three dimensional crystal structure of SSU (PDB: 2AVY)34, 180° rotation shown. Colors are in as (b) and several r-proteins are labelled for reference. Solvent surface is shown (see Supplementary Fig. 4b for interface surface).
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Figure 4: Multiple pathways for ribosomal proteins addition to the three intermediates(a) Spectral counts of r-proteins bound to intermediates are compared to mature ribosomes purified with tag at position 86M (see Methods, Supplementary Data Set 2) and relative r-protein levels are calculated as percentages [biological replicates (n = 3) and technical replicates (n = 2)]. R-protein percentages of each intermediate are binned into 4 groups and plotted as heat maps on the three-dimensional crystal structure of SSU (PDB: 2AVY)34. Solvent side is shown (see Supplementary Fig. 4a for interface side). (b) Hierarchical clustering analysis of r-proteins bound to the 105L, 11L, and 20T tagged intermediates. Relative protein abundance (RPA) for each r-protein present in each of the three SSU intermediates (but not mature SSUs) is calculated as described in Methods. Different shades of green indicate the levels of occupancy by a specific r-protein among the intermediates. The darker the green color, the more abundant the r-protein is bound in that intermediate as compared to the other intermediates. The r-proteins are clustered into three major groups, marked as I, II and III. (c) RPA of r-proteins associated with three intermediates is plotted on the three dimensional crystal structure of SSU (PDB: 2AVY)34, 180° rotation shown. Colors are in as (b) and several r-proteins are labelled for reference. Solvent surface is shown (see Supplementary Fig. 4b for interface surface).

Mentions: To further investigate differences between the 17S rRNA containing RNPs, we examined r-protein association by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectral counting (Fig. 4, Supplementary Fig. 4). We used mature ribosomes, purified with an MS2 tag in the mature 16S rRNA (86M), as a normalization standard for r-protein association (Supplementary Data Set 2), allowing direct comparison of r-protein levels in intermediates to mature SSUs. No r-protein was found in any of the three 17S rRNA containing RNPs, at levels observed in mature SSUs (Fig. 4a, Supplementary Fig. 4a). R-protein, S4, which binds to the 5′ domain of 16S rRNA, corresponding to the SSU body, had approximately the same occupancy in all three intermediates. Three r-proteins, S2, S3 and S21 were highly underrepresented in all intermediates as compared to mature SSUs (Fig. 4a, ≤38%). 105L tagged rRNPs had the lowest total r-protein association compared to the other two pre-SSUs and mature SSUs. 11L and 20T tagged rRNP had distinct r-protein association. Thus, distinct r-protein composition corroborates the different conformations observed in probing experiments and further supports that each affinity purified intermediate is distinct.


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)

Multiple pathways for ribosomal proteins addition to the three intermediates(a) Spectral counts of r-proteins bound to intermediates are compared to mature ribosomes purified with tag at position 86M (see Methods, Supplementary Data Set 2) and relative r-protein levels are calculated as percentages [biological replicates (n = 3) and technical replicates (n = 2)]. R-protein percentages of each intermediate are binned into 4 groups and plotted as heat maps on the three-dimensional crystal structure of SSU (PDB: 2AVY)34. Solvent side is shown (see Supplementary Fig. 4a for interface side). (b) Hierarchical clustering analysis of r-proteins bound to the 105L, 11L, and 20T tagged intermediates. Relative protein abundance (RPA) for each r-protein present in each of the three SSU intermediates (but not mature SSUs) is calculated as described in Methods. Different shades of green indicate the levels of occupancy by a specific r-protein among the intermediates. The darker the green color, the more abundant the r-protein is bound in that intermediate as compared to the other intermediates. The r-proteins are clustered into three major groups, marked as I, II and III. (c) RPA of r-proteins associated with three intermediates is plotted on the three dimensional crystal structure of SSU (PDB: 2AVY)34, 180° rotation shown. Colors are in as (b) and several r-proteins are labelled for reference. Solvent surface is shown (see Supplementary Fig. 4b for interface surface).
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Related In: Results  -  Collection

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Figure 4: Multiple pathways for ribosomal proteins addition to the three intermediates(a) Spectral counts of r-proteins bound to intermediates are compared to mature ribosomes purified with tag at position 86M (see Methods, Supplementary Data Set 2) and relative r-protein levels are calculated as percentages [biological replicates (n = 3) and technical replicates (n = 2)]. R-protein percentages of each intermediate are binned into 4 groups and plotted as heat maps on the three-dimensional crystal structure of SSU (PDB: 2AVY)34. Solvent side is shown (see Supplementary Fig. 4a for interface side). (b) Hierarchical clustering analysis of r-proteins bound to the 105L, 11L, and 20T tagged intermediates. Relative protein abundance (RPA) for each r-protein present in each of the three SSU intermediates (but not mature SSUs) is calculated as described in Methods. Different shades of green indicate the levels of occupancy by a specific r-protein among the intermediates. The darker the green color, the more abundant the r-protein is bound in that intermediate as compared to the other intermediates. The r-proteins are clustered into three major groups, marked as I, II and III. (c) RPA of r-proteins associated with three intermediates is plotted on the three dimensional crystal structure of SSU (PDB: 2AVY)34, 180° rotation shown. Colors are in as (b) and several r-proteins are labelled for reference. Solvent surface is shown (see Supplementary Fig. 4b for interface surface).
Mentions: To further investigate differences between the 17S rRNA containing RNPs, we examined r-protein association by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectral counting (Fig. 4, Supplementary Fig. 4). We used mature ribosomes, purified with an MS2 tag in the mature 16S rRNA (86M), as a normalization standard for r-protein association (Supplementary Data Set 2), allowing direct comparison of r-protein levels in intermediates to mature SSUs. No r-protein was found in any of the three 17S rRNA containing RNPs, at levels observed in mature SSUs (Fig. 4a, Supplementary Fig. 4a). R-protein, S4, which binds to the 5′ domain of 16S rRNA, corresponding to the SSU body, had approximately the same occupancy in all three intermediates. Three r-proteins, S2, S3 and S21 were highly underrepresented in all intermediates as compared to mature SSUs (Fig. 4a, ≤38%). 105L tagged rRNPs had the lowest total r-protein association compared to the other two pre-SSUs and mature SSUs. 11L and 20T tagged rRNP had distinct r-protein association. Thus, distinct r-protein composition corroborates the different conformations observed in probing experiments and further supports that each affinity purified intermediate is distinct.

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