Ribosome biogenesis is a fundamental and multistage process. The basic steps of ribosome assembly are the transcription, processing, folding, and modification of rRNA; the translation, folding, and modification of r-proteins; and consecutive binding of ribosomal proteins to rRNAs. Ribosome maturation is facilitated by biogenesis factors that include a broad spectrum of proteins: GTPases, RNA helicases, endonucleases, modification enzymes, molecular chaperones, etc. The ribosome assembly factors assist proper rRNA folding and protein–RNA interactions and may sense the checkpoints during the assembly to ensure correct order of this process. Inactivation of these factors is accompanied by severe growth phenotypes and accumulation of immature ribosomal subunits containing unprocessed rRNA, which reduces overall translation efficiency and causes translational errors. In this review, we focus on the structural and biochemical analysis of the 30S ribosomal subunit assembly factors RbfA, YjeQ (RsgA), Era, KsgA (RsmA), RimJ, RimM, RimP, and Hfq, which take part in the decoding-center folding.

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.

Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. TheEscherichia coli lysCriboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests thatlysCgene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that thelysCriboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation oflysCexpression. Together with previous studies, our work suggests thatlysCexpression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.

Small RNAs (sRNAs) controlling gene expression by imperfect base-pairing with mRNA(s) are widespread in bacteria. They regulate multiple genes, including genes involved in iron homeostasis, through a wide variety of mechanisms. We previously showed that OmrA and OmrB sRNAs repress the synthesis of the Escherichia coli FepA receptor for iron–enterobactin complexes. We now report that five additional sRNAs, namely RprA, RybB, ArrS, RseX and SdsR, responding to different environmental cues, also repress fepA, independently of one another. While RprA follows the canonical mechanism of pairing with the translation initiation region, repression by ArrS or RseX requires a secondary structure far upstream within the long fepA 5′ untranslated region. We also demonstrate a dual action of SdsR, whose 5′-part pairs with the fepA translation initiation region while its 3′-end behaves like ArrS or RseX. Strikingly, mutation analysis shows a key role for the loops of these sRNAs’ intrinsic terminators in the regulation. Furthermore, regulation depends on both the Hfq chaperone and the RNase E endonuclease. Overall, our data strongly suggest that FepA levels must be tightly controlled under a variety of conditions and highlight the diversity of mechanisms that underly the regulation of gene expression by sRNAs in bacteria.