The ribosomal maturation factor P from Mycobacterium smegmatis facilitates the ribosomal biogenesis by binding to the small ribosomal protein S12

Abstract

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli. Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis. However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae. Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N-and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections. by guest on July 10, 2020 http://www.jbc.org/ Downloaded fromFigure 4. The efficiency of in vivo ribosomal biogenesis of MSMEG_2624 mutants.A, sucrose gradient centrifugation of the WT M. smegmatis, the MSMEG_2624 knockout strain, the MSMEG_2624 knockout with the vector that expresses MSMEG_2624, and the MSMEG_2624 knockout that carries the empty vector. The vertical yellow bar marks the fraction further characterized using MS in B. B, The bar plot shows the ratio of S12/S4 in the 30S subunit of WT, the MSMEG_2624 deletion mutant strain, and the MSMEG_2624 knock-out strain with the vector that expresses MSMEG_2624. C, sucrose gradient centrifugation shows the ribosome profile of the MSMEG_2624 mutants which affects binding with RpsL (⌬P90-D93, shown in green), and which does not (P95G, shown in red). The controls of empty vector and empty vector ϩ WT are shown in cyan and purple. RimP facilitates ribosomal biogenesis by binding to S12

A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

“…Extending the previously described role of RimP in promoting S12 binding (29–31), our results also indicate a broader role in 30S subunit assembly where RimP delays h44 positioning on the front of the 30S subunit, thus exposing the RsmA binding site, and may even pre-associate with RsmA to facilitate its recruitment to the subunit. Moreover, by keeping the upper part of h44 and its linker regions disordered, RimP maintains an open unstructured channel in the 30S neck region for the 16S 3′-end to swap.…”

“…In the presented series of ribosomal complexes (Figure 1), the first assembly factors to bind the 30S subunit are RimP and RsmA. While RsmA, from the KsgA/Dim1 protein family, is a universally conserved rRNA methylase that modifies residues A1518 and A1519 in h45 (28), RimP is essential for survival under stress conditions in Mycobacterium (5, 29). Among all factors studied here, RimP is the most versatile and is seen in four assembly states (state A to D ) bound on the front of the 30S subunit (blue in Figure 1).…”

“…Among all factors studied here, RimP is the most versatile and is seen in four assembly states (state A to D ) bound on the front of the 30S subunit (blue in Figure 1). During ribosome biogenesis, RimP is known to promote binding of the r-protein uS12 (29–31). Accordingly, our cryo-EM data shows it to interact directly with uS12 by forming an intermolecular β-sheet via anti-parallel pairing between uS12 strand β1 and RimP strand β3 (Figure 3A).…”

Identification of 40S ribosomal protein S8 as a novel biomarker for alcohol‑associated hepatocellular carcinoma using weighted gene co‑expression network analysis

“…The small ribosomal family proteins are important structural components of the ribosome, which serves a key role in protein synthesis (20). The functional biology and related molecular mechanisms of small ribosomal proteins have been widely investigated (21).…”

“…(A and B) Scale independence and mean connectivity of various soft-threshold values (β). The red number indicates the different soft threshold values(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), while the red lines indicates the cut-off values selected, as the scale independence >0.85. (C) Gene sets (black circles) with corresponding log10 (connectivity) and log10 P-value (connectivity) when the scale-free topology is set as β=14.…”