A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

Thank you for sending your work entitled “A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli” for consideration at eLife. Your article has been favorably evaluated by James Manley (Senior editor), a Reviewing Editor, and two other reviewers, both of whom, Harry Noller and Peter Moore, have agreed to reveal their identity.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The two reviewers praised your paper as exceptional. Their assessment is as follows:

1) This paper addresses one of the most complex and challenging problems in biology – the mechanism of assembly of ribosomes in vivo. The authors use a combined mass spec/EM approach in an impressive technical tour de force. They first analyze 30S subunit assembly intermediates obtained from sucrose gradient fractions from wild-type cells, correlating the absence of specific ribosomal proteins with characteristic features of EM images of immature particles. They then use their approach to compare wild-type assembly with that of cells carrying mutations that affect 30S assembly. A major finding is that the ribosome assembly factor rimP appears to be critical for formation of the central pseudoknot of 16S rRNA and assembly of its associated ribosomal proteins. This is an outstanding paper that advances our understanding of in vivo ribosome assembly and demonstrates the use of a powerful new approach to studying the assembly of complex cellular particles.

2) This manuscript reports the outcome of an extensive series of experiments the authors have performed to characterize the pathway of ribosome assembly in E. coli in vivo. The powerful technology they have used to do this work has been perfected by them over a period of several years, and is likely to prove useful for others interested in the assembly of similarly complex macromolecular assemblies. This study goes a long way towards proving that RimP, a gene product long known to be involved in ribosome assembly, plays an important role in ensuring that the central pseudoknot in 16S rRNA forms properly during assembly.

The insights obtained by the authors reveal the unique power of the experimental system they have devised, and represent a significant advance in our understanding of ribosome assembly, a physiological process of the utmost importance biologically.

Minor comments:

1) The “degradation” observed may be due to the presence of endogenous RNase H.

2) The color coding used in Figure 1C was counter-intuitive. It took quite some time to “read” the protein levels as intended. The authors might want to consider using a more usual “heat map” range (ramping, say, from blue to red) to more clearly show the abundance values, which are very critical to the punch line of the paper.

3) I was puzzled by the absence of signal for protein S17, which was only alluded to in a cryptic comment toward the end. Perhaps this has already been addressed in a previous paper?

1) The “degradation” observed may be due to the presence of endogenous RNase H.

This is one possibility, although we observed increasing amounts of degradation over time and in the absence of RNase inhibitor, suggesting that the degradation was mainly due to non-specific RNase activity. We have amended the text as follows to explain our interpretation of the degradation: “The 3’-domain particles likely result from non-specific cleavage of the exposed central PK region in Group II particles by contaminating RNases in the sample used for affinity purification. Efforts were made to limit sample degradation using RNase inhibitors, with limited success, further indicating the extent of rRNA exposure in the ΔrimP intermediates.”

Figure legend for Figure 6–figure supplement 1D now reads: “In sample 2, degradation was limited by the addition of RNasin (Promega) and reducing the amount of time for sample preparation.”

2) The color coding used in Figure 1C was counter-intuitive. It took quite some time to “read” the protein levels as intended. The authors might want to consider using a more usual “heat map” range (ramping, say, from blue to red) to more clearly show the abundance values, which are very critical to the punch line of the paper.

We have changed the coloring of Figure 1C, 4C and Figure 4–figure supplement 1C to ramp from red to white to blue (0-1 relative protein levels).

3) I was puzzled by the absence of signal for protein S17, which was only alluded to in a cryptic comment toward the end. Perhaps this has already been addressed in a previous paper?

We generally observe very few peptides for S17, and the peptides that we do detect often have poor isotope distribution fits. The poor fits cannot be attributed to very low levels of S17 in experimental samples, because the problem occurs for both the experimental ¹⁴N peptides from intact ribosomes and the reference ¹⁵N peptides. In these datasets, we could not unambiguously assign the poorly fit S17 peptides, and so they were excluded from our analysis. We have added the following sentences to the text to clarify this lack of data: “Peptides were detected for all r-proteins with the exception of later binding proteins with very low abundance in fraction 1 (S2, S3, S13, S19, S21) and S17 for fractions 1, 4 and 5 (Figure 1C). The isotope distribution fits for S17 peptides are often poor for both the experimental and reference sample, preventing unambiguous assignment of these peptides and necessitating their exclusion from the qMS analysis.”