Directed hydroxyl radical probing using complexes containing Fe(II)-S20 and 16S rRNA reveal aspects of rRNA domain organization and 30S subunit assembly. The results of the directed hydroxyl radical probing in the minimal Fe(II)-S20/16S rRNA complexes and fully assembled subunits correlate well with the relative positions of the derivatized residues in S20 ( and ) and the cleavage sites and their intensity correlate well with the placement of the respective probe in the structure of r-protein S20 and of the 30S subunit (, and ). Our data are consistent with a substantial folding and architectural organization of the 5′ domain early in assembly (see 9; 13; 14; 15; 16
) and with the final alignment of helix 44 occurring later in the assembly cascade9; 11
. These results, taken together with our earlier work suggest that there are different means by which domains or elements of 16S rRNA are organized.
Overall, our data from Fe(II)-S20 probing in 30S subunits is in good agreement with the structures of 30S subunits3,4
. A large majority of the cleavage sites are within the accepted 40 Å range for tethered cleavage from r-proteins32
. However, there are nuances in the data, such as cleavage of helical faces that do not absolutely agree with the relative positioning to the S20 probing sites, which need to be addressed. A plausible explanation for data such as these is that the minimal RNPs and 30S subunits are dynamic and can adopt multiple conformations, as has been observed crystallographically4, 32
. Thus, it seems likely that in our probing experiments we are examining an ensemble of 30S subunit or RNP structures and thus our experiments may allow us to access more conformations than could be allowed in a crystal lattice. This idea is not unprecedented as differences in solvent accessibility observed in crystal structures and reactivity of rRNA to hydroxyl radicals has been observed34
. In this work, it was hypothesized, after several other possible hypotheses were examined and refused, that these differences were due to the existence of different conformations in solution and that the crystal structure may represent only one of these possible conformations. It should also be mentioned that the addition of our BABE-linker could change some local conformations or dynamics and thereby result in some of the subtle discrepancies between our data and what would be predicted from the crystal structures. Nonetheless, the level at which our data correlates with structural information is striking and sufficient to suggest that S20 is binding at a similar site in the minimal RNPs, the 30S subunits studied herein and in the subunits studies by crystallography, yet differences in overall architecture, dynamics and details of the interactions are possible.
Three of the four derivatized Fe(II)-S20 proteins have essentially the same cleavage patterns in the 5′ domain of 16S rRNA, in the binary particle and fully assembled 30S subunit (see ). For Fe(II)-C23, -C49 and -C57, the 5′ domain cleavage patterns only differ slightly in these rather distinct complexes. In contrast, when Fe(II)-C14-S20 is used as a probe the cleavage patterns in the minimal complex and the 30S subunit are quite distinct although there is an underlying similarity ( and ). Many of the cleavage sites from Fe(II)-C14-S20 observed in the small subunit are present in the binary complex however the intensity of cleavage is significantly weaker. Verification of these data (the weak cleavage sites) required much additional repetition of probing experiments and primer extension from multiple, overlapping primers. Base-specific footprinting demonstrated that this derivatized protein does bind 16S rRNA under the isolation and purification conditions used herein and thus the “lack” of cleavage is not due simply to the absence of this protein in the minimal complexes. Some of the differences observed when probing is initiated from position 14 of the r-protein can be explained by its location in S20 and in the 30S subunit, and by the structural changes that likely take place in S20 upon further assembly (i.e. binding of r-protein S17). Amino acid 14 of r-protein S20 is located on its N-terminal helix, the longest helix of the three that compose S20 (). This portion of the N-terminal helix is not part of the helical bundle, but is somewhat extended and isolated from other protein elements. Unlike the other primary binding r-proteins (S4, S7, S8, S15 and S17) whose structures in the unliganded state have been determined by NMR, crystallography or both17–25
, the structure of S20 has not been determined in the free state. Moreover, it has been shown that free S20 has only about 36% helical content26
, compared to its structure in the 30S subunit where it is almost completely helical4
. It is possible that changes in S20 conformation particularly in the N-terminal helix, upon binding of additional r-proteins such as S17, could explain changes in the cleavage patterns. The N-terminal helix of r-proteins S20 in the structure of the 30S subunit is enclosed in a channel formed by the tertiary interaction between helix 8 and helix 14. The cleavage sites in helix 11, 13 and 14 appear for Fe(II)-C14-S20 only after binding of S17. We can envision that binding of S17 helps the interaction between helix 14 and helix 8, and both the r-protein S20 and the rRNA surrounding it adopt the appropriate conformation. Thus, a combination of the structure and dynamics of the N-terminal helix of S20 and the 16S rRNA surrounding it, may account for the lower intensity of cleavage in the minimal complex from position C14 and further experiments will be required to fully understand these observations.
R-proteins S16 and S17 appear to play interesting roles in altering the cleavage patterns from Fe(II)-C14-S20. The cleavage that is observed in helix 5 of 16S rRNA in a complex containing Fe(II)-C14-S20 and other r-proteins including S17 is almost completely diminished upon addition of S16 and this cleavage is absent in the 30S subunit. While S16 is proximal to helix 5 it seems unlikely that the reduced cleavage is direct since S16 does not contact helix 5 in the 30S subunit3,4
. Helix 15 is sandwiched between S16 and helix 5 and S16 is in direct contact with helix 15 (). Thus, it is possible that upon S16 association, helix 15 is organized and packs against helix 5 thus resulting in the changes observed in this work. The effect of S16 on helix 5 cleavage would be indirect, and this is also likely for the majority of changes associated with S17 binding given the large distance from S17 to many of the sites. These observations suggest that there are concerted 16S rRNA folding events that are mediated by r-proteins resulting in a hierarchical assembly cascade.
The results of cleavage when Fe(II) is tethered to position C49 of S20 are almost the opposite of what was just mentioned for Fe(II)-C14-S20. Very similar patterns of cleavage are observed when probing is initiated from Fe(II)-C49-S20 in both the binary and fully assembled complexes. Amino acid 49 is very near the junction of the helical bundle of S20 where the three helical elements come together and is the furthest probing position from C14. It is plausible that the N-terminal helix where C14 is localized is unstructured, as explained above. Nevertheless it is possible that the helical region near amino acid 49 of S20 is well formed upon interaction with 16S rRNA and that its environment is not changed significantly after formation of the binary complex. Additionally, many of the sites that are cleaved from Fe(II)-C49-S20 are unique to this tethering position and/or they are more on the periphery of 16S rRNA as it is folded in the 30S subunit4
Additionally, given the strength of the cleavages in helix 44 from Fe(II)-C49-S20 it is possible that they are less sensitive to changes in helix 44 alignment and positioning. It is interesting that cleavage of helix 44 is so strong only from C49 in the minimal complex, although in the 30S subunit structure amino acids 23 and 57 are as close to these elements. Interestingly in the 30S subunit there are not significant differences in the distance between the three different probing sites and the most remote or proximal cleavage sites in helix 44: for C23 the range is 8–43 Å, for C49 it is 14–39 Å and for C57 it is 11–42 Å. Thus proximity as measurable in the available 30S subunit structure cannot explain the differences in probing results. Again, these differences could be due to strength of cleavage, S20 structure and folding, quenching from other 16S rRNA elements and/or dynamics. This portion of r-protein S20 and the elements of the rRNA cleaved from it may fold earlier in assembly or simply be more accessible and therefore fewer differences in cleavage patterns are observed between minimal and fully assembled RNPs.
Significant differences are observed in the cleavage patterns in helix 44 for binary complexes and the 30S subunit for three of the derivatized proteins Fe(II)-C14-S20, Fe(II)-C23-S20 and Fe(II)-C57-S20, while for Fe(II)-C49-S20 there are small variations. Most intriguing are the results obtained when Fe(II)-C23-S20 and Fe(II)-C57-S20 are used as probes, since significant differences in the cleavage patterns between the binary complexes and 30S subunits were observed. As mentioned above for these two probing positions only small differences in intensity were detected in the 5′ domain (see and ) suggesting that derivatization of these positions does not interfere with S20 binding, which is consistent with footprinting data (data not shown). Residues 23 and 57 of r-protein S20 are located close to helix 44 in the fully assembled 30S subunit (), and four hydrogen bonds between S20 and 16S rRNA are present spatially close to these derivatized residues (Arg28-A1437, Lys32-G1439, Ser25-G1459, Thr39-G1458)4; 27
. In the binary complexes containing Fe(II)-C23-S20 or Fe(II)-C57-S20, little cleavage was observed in helix 44, while three and four cleavage sites, respectively were observed for the 30S subunit ( and ). Therefore, we cannot unequivocally rule out the possibility that the derivatization of these S20 residues interferes with the orientation of the penultimate stem, and that in the fully assembled subunit this interference is overcome. However, the cleavage patterns from Fe(II)-C49-S20 similar for both RNPs studied here (see ) and the S20-specific footprints observed in helix 44 in the S20/16S rRNA complexes do not support this hypothesis. Also, similar late docking of helix 44 was observed in tethered probing experiments using Fe(II)-S1510
, which does not directly contact helix 444
. Thus, it seems likely that the penultimate stem does not interact fully with S20 until late in assembly. Distance measurements within the 30S subunit support this idea. Position C23 of S20 is only 8 Å from some of its cleavage sites in helix 44 but these sites are not cleaved in any of the minimal RNPs tested. Similarly, the distance from C57 and some helix 44 cleavage sites is on the order of 12 Å. These data suggest that either S20 or helix 44, or both, are in an alternative environment or conformation in the minimal RNPs as compared with the fully assembled subunit. The results presented in this manuscript and the previous ones using Fe(II)-S15 as a probe 10
suggest that the positioning of this functionally important rRNA element can occur late in 30S subunit assembly.
Based on our data and that available in the literature 10; 15
, we suggest that in the binary complex of S20 and 16S rRNA interaction is mainly between the 5′ domain of 16S rRNA and S20, with minimal interaction with the penultimate stem. The footprints observed in the minimal complex of S20/16S rRNA by chemical probing1
could probably arise from base pairing or RNA-RNA interactions since the protected nucleotides are not directly involved in 16S rRNA/S20 interactions in the fully assembled 30S subunit4; 27
. Somewhat later in assembly, in the presence of other r-proteins, the interaction between helix 44 and S20 becomes complete and thus their interaction aids in positioning of this important rRNA element. The 5′ domain seems to be fairly well organized even in the absence of r-proteins15
and binding of S20, may aid this rRNA domain in adopting an architecture very similar to the one in the fully assembled 30S subunit. In contrast, the other three domains of 16S rRNA appear to require the presence of r-proteins (in various degrees) to achieve their functional structure. In the minimal, binary Fe(II)-S15/16S rRNA complex the number of cleavage sites was small and localized to a small region in the central domain of 16S rRNA10; 11
. Probing from Fe(II)-S15 in the fully assembled 30S subunit yielded significantly more cleavage sites throughout the central domain and extending to others including the 3′ minor domain. Many of these cleavage sites were present in RNPs of higher complexity. The 3′ minor domain, which interacts mainly with r-protein S20, seems to adopt its final structure late in the assembly. The results presented in this work highlight the complexity of RNP assembly, particularly that of the small ribosomal subunit.