Structures of Mycobacterium smegmatis 70S ribosomes in complex with HPF, tmRNA, and P-tRNA

Abstract

Ribosomes are the dynamic protein synthesis machineries of the cell. They may exist in different functional states in the cell. Therefore, it is essential to have structural information on these different functional states of ribosomes to understand their mechanism of action. Here, we present single particle cryo-EM reconstructions of the Mycobacterium smegmatis 70S ribosomes in the hibernating state (with HPF), trans-translating state (with tmRNA), and the P/P state (with P-tRNA) resolved to 4.1, 12.5, and 3.4 Å, respectively. A comparison of the P/P state with the hibernating state provides possible functional insights about the Mycobacteria-specific helix H54a rRNA segment. Interestingly, densities for all the four OB domains of bS1 protein is visible in the hibernating 70S ribosome displaying the molecular details of bS1-70S interactions. Our structural data shows a Mycobacteria-specific H54a-bS1 interaction which seems to prevent subunit dissociation and degradation during hibernation without the formation of 100S dimer. This indicates a new role of bS1 protein in 70S protection during hibernation in Mycobacteria in addition to its conserved function during translation initiation.

Ribosome profiling inMycobacterium tuberculosisreveals robust leaderless translation

“…RafS is homologous to MSMEG_3935, which contains an S30AE domain and a ribosome-binding domain and has been shown to bind mycobacterial ribosomes under conditions of hypoxic stress (Trauner et al, 2012). Biochemical characterisation of RafS including structural studies of this protein bound to mycobacterial ribosomes indicates a mycobacterial-specific interaction which seems to prevent subunit dissociation and degradation during hibernation without the formation of 100S dimer (Mishra et al, 2018). Our data clearly show that translation of leaderless transcripts increases during nutrient starvation, but there was no evidence that this is due to a difference in the TE between different types of transcripts under different conditions.…”

Stress response as implemented by hibernating ribosomes: a structural overview

“…Alternatively, one cannot exclude the possibility that in the cell, HPF long is a monomer and upon binding to the ribosome, dimerization occurs via homodimerization of the HPF long -CTD. The presence of HPF long does not necessarily result in ribosome dimer formation, as demonstrated by the structures of ribosomes from Mycobacterium smegmatis [37,38] (which is frequently used as a model of Mycobacterium tuberculosis) and the spinach chloro-ribosome [35], in complex with HPF long . Both organisms carry HPF long ; but ribosomal dimers have not been reported in these species.…”

“…In the chloroplasts, bS1c is tightly associated with the chloro-ribosome and M. smegmatis has an rRNA extension, H54a, a unique feature of mycobacteria, which seems to bind to bS1 and stabilize it, as well as H54a itself. Sequence comparison of HPF long -CTD from M. smegmatis, M. tuberculosis, S. aureus, and L. lactis shows that one out of the five conserved residues proposed to be involved in the formation of 100S dimers is not conserved in Mycobacteria [32,37]. However, it is not clear that this is the reason that prevents 100S dimer formation in M. smegmatis.…”

“…Alternatively, one cannot exclude the possibility that in the cell, HPF long is a monomer and upon binding to the ribosome, dimerization occurs via homodimerization of the HPF long -CTD. Sequence comparison of HPF long -CTD from M. smegmatis, M. tuberculosis, S. aureus, and L. lactis shows that one out of the five conserved residues proposed to be involved in the formation of 100S dimers is not conserved in Mycobacteria [32,37]. Both organisms carry HPF long ; but ribosomal dimers have not been reported in these species.…”

“…2A). The presence of HPF long does not necessarily result in ribosome dimer formation, as demonstrated by the structures of ribosomes from Mycobacterium smegmatis [37,38] (which is frequently used as a model of Mycobacterium tuberculosis) and the spinach chloro-ribosome [35], in complex with HPF long . Alternatively, one cannot exclude the possibility that in the cell, HPF long is a monomer and upon binding to the ribosome, dimerization occurs via homodimerization of the HPF long -CTD.…”