Post-transcriptional modifications and temperature as determinants of ribosomal subunit maturation

Abstract

The biosynthesis of ribosomes (ribosome assembly) represents one of the most energy-demanding and tightly regulated process in bacteria such as Escherichia coli. In vivo, this complex pathway is coordinated by a network of assembly factors that modify and guide rRNA folding, mediate ribosomal protein incorporation, and ensure the formation of translationally competent ribosomal subunits. These assembly factors are essential for maintaining protein synthesis and cellular fitness, particularly under changing environmental conditions. However, the molecular mechanisms governing the coordinated action of ribosome assembly factors in vivo, especially their functional interplay, remain poorly understood. In addition, the parameter temperature is of critical importance, since RNA tends to adopt misfolded conformations under reduced thermal energy conditions. In this thesis, a comprehensive set of 30S and 50S assembly factors was examined using an antisense RNA-mediated protein depletion strategy, allowing direct comparison of phenotypic effects and ribosome assembly defects. Growth and polysome profile analysis revealed temperature-dependent phenotypes, with most depletion strains displaying cold sensitivity (e.g. ObgE), whereas some exhibited assembly defects at elevated temperatures (e.g. EngA). It is exceptional that these two essential GTPases ObgE and EngA, both contributing to the formation of the peptidyl transferase center (PTC), showed inverse temperature dependencies. Although the thermodynamic and structural details of the arrested precursors remain unclear, it was remarkable to observe that the precursor accumulating under ObgE depletion in the cold could be converted into a mature 50S subunit by providing thermal energy. This establishes a rational that, in some cases, thermal energy can substitute for assembly factor-mediated remodeling. Furthermore, ObgE depletion resulted in the selective degradation of immature 50S particles. Mass spectrometry and biochemical analyses revealed the recruitment of ribonucleases RNase E and RNase R, suggesting a novel ribonuclease-dependent quality control pathway in 50S ribosome assembly. This surveillance mechanism ensures ribosome homeostasis by recycling incomplete complexes and preserving the translationally active ribosome pool. Complementary super-resolution microscopy demonstrated that ribosome assembly in E. coli is spatially organized within condensate-like microenvironments, reminiscent of the eukaryotic nucleolus. Perturbations in ribosome assembly altered the VI chromosomal morphology and in case of ObgE depletion, led to the co-localization of the RNA helicase DeaD with ribosomal foci, suggesting a distinct spatial compartmentalization of bacterial ribosome assembly. The findings of this thesis collectively propose a model in which temperature, rRNA modification, and energy-dependent assembly factors collectively ensure efficient and accurate ribosome assembly. By integrating thermodynamic plasticity, active enzymatic remodeling, and ribonuclease-mediated quality control, and spatiotemporal organization, this work reveals how E. coli dynamically balances ribosome production with environmental and physiological demands.