Ribosomes are large ribonucleoprotein assemblies that conduct the process of translation of the genetic code. They are composed of two asymmetric subunits, small and large, which associate through a network of intermolecular interactions. Many antibiotics, which are widely used in the treatment of bacterial infections, interfere with protein synthesis. A large number of them bind to the small ribosomal subunit [1
] and block proper ribosome function either by hindering the decoding process or by inhibiting the functional conformational changes of the ribosome [2
]. The understanding of interactions governing the assembly mechanism is of great importance because recently it has also been found that the aminoglycoside antibiotics inhibit not only the translation itself but also the formation of the small subunit [3
In bacteria, the small and large subunits are named, according to their sedimentation coefficients, 30S and 50S, respectively. The 30S subunit, which is the subject of this study, consists of the 16S ribosomal RNA (16S rRNA), and 21 proteins which are labeled S1, S2,
S21. During ribosome activity, messenger RNA and transfer RNA molecules bind to the small subunit. The main role of the small subunit is to maintain translation fidelity by assuring for correct decoding. In the early 1970s, it was found that the Escherichia coli
small ribosomal subunit can reassemble in vitro from the 16S rRNA and a mixture of the 30S proteins [4
]. Such reassembly produces an active 30S particle, and these experiments revealed that subunit complexation is a sequential and ordered process. The proteins were classified as primary, secondary, or tertiary binders, depending on their ability to bind alone or only in the presence of other proteins. The experimentally derived assembly order map is presented in . Since then, the pathway and the mechanism of the assembly have been of significant interest (for review see [6
]), however, many details of this process still remain unclear.
The 30S Subunit In Vitro Experimental Assembly Map for E. coli
Apart from the “assembly order map” of Nomura and coworkers [4
], a “kinetic assembly map” was also determined [7
]. The kinetics-based map classifies the proteins as early, middle, middle-late, and late binders, and suggests that the assembly of proteins proceeds roughly from the 5′, through central, to the 3′ domain of 16S rRNA even though in vitro this process is not coupled with the temporal order of transcription of the proteins. These two maps (assembly order and assembly kinetics) serve as a model of the ordered assembly of E. coli
30S subunits. Because all the information needed for the small subunit assembly is present in the 16S rRNA and protein components, it should be possible to study this process based on the crystallographic structures of the 30S ribosomal complex.
Apart from the vast amount of experimental approaches to study the association of proteins with 16S rRNA, theoretical modelling approaches of the 30S subunit assembly have not been numerous. Coarse-grained Monte Carlo simulations have been performed to assess the change in fluctuations upon binding of proteins in the 3′ domain assembly [8
] and to predict the contributions of each of the proteins to the organization of the binding sites for the sequential proteins in the S7 pathway. Recently, a similar coarse-grained force field was applied in molecular dynamics simulations of the small subunit assembly [9
]. However, these two studies focus more on the 16S rRNA conformational changes due to the binding events than on the energetics of the 30S assembly. To account for the latter, we have previously applied an implicit solvent Poisson-Boltzmann model to study the relative binding free energies of 30S proteins to 16S rRNA [10
]. The Poisson-Boltzmann all-atom investigation, even though giving encouraging results, was performed on a single 30S subunit configuration and was somewhat sensitive to applied parameters, such as the dielectric constant of the subunit and the placement of the dielectric boundary between the 30S molecule and implicit solvent.
The biggest drawback of all these approaches is that they are time-consuming thus are not applicable to the several thousands of configurations we have made for this study. This huge set of configurations is however necessary to deduce interdependencies in a comprehensive fashion. Therefore, we decided to base this work on a computationally faster but still accurate approach which can focus both on the changes in energetics and in fluctuations. Our model is based on the self-consistent pair contact probability approximation (SCPCP) by Micheletti et al. [11
]. The SCPCP has several advantages over other coarse-grained and computationally fast methods: a) while it is based on the fluctuations of residues, it can—in contrast to elastic network models—break contacts between residues, b) the SCPCP free energy also includes entropies that are not accessible by methods that compute binding energies as a sole sum of knowledge based interaction strengths. The latter cannot provide for long-range influences, which we found to be relevant in the assembly (see Results
We calculated the dependencies of protein binding to 16S rRNA for the Thermus thermophilus small ribosomal subunit and were able to reproduce in many aspects the E. coli assembly map as well as predict the differences of assembly between those two bacteria. We were able to identify key proteins most important in mutual stabilization. In addition, we propose a mechanism of binding for the THX-peptide. In future work, the model may be easily applied to the large ribosomal subunit for which a detailed experimental map of binding is not yet available.
The paper is organized as follows; the Results present the interdependencies of protein binding for the T. thermophilus bacterium derived from computational experiments for the removal from the 30S complex of one or two proteins at a time. The similarities and differences with the E. coli assembly map are discussed. The computational model and the parameterization are presented in the Materials and Methods section.