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Functional Analysis of BipA in E. coli Reveals the Natural Plasticity of 50S Subunit Assembly.

ABSTRACT: BipA is a conserved translational GTPase of bacteria recently implicated in ribosome biogenesis. Here we show that Escherichia coli ΔbipA cells grown at suboptimal temperature accumulate immature large subunit particles missing several proteins. These include L17 and L17-dependent binders, suggesting that structural block 3 of the subunit folds late in the assembly process. Parallel analysis of the control strain revealed accumulation of nearly identical intermediates, albeit at lower levels, suggesting qualitatively similar routes of assembly. This came as a surprise, because earlier analogous studies of wild-type E. coli showed early binding of L17. Further investigation showed that the main path of 50S assembly differs depending on conditions of growth. Either supplementation of the media with lysine and arginine or suboptimal temperature appears to delay block 3 folding, demonstrating the flexible nature of the assembly process. We also show that the variant BipA-H78A fails to rescue phenotypes of the ΔbipA strain, indicating a critical role for GTP hydrolysis in BipA function. In fact, BipA-H78A confers a dominant negative phenotype in wild-type cells. Controlled production of BipA-H78A causes accumulation of 70S monosomes at the expense of polysomes, suggesting that the growth defect stems from a shutdown of translation.

SUBMITTER: Gibbs MR

PROVIDER: S-EPMC7502552 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Functional analysis of BipA in E. coli reveals the plasticity of 50S ribosome assembly

Project description:BipA is a conserved translational GTPase that resembles elongation factor EF-G and 30S assembly factor LepA. Recent evidence suggests that BipA functions in 50S subunit assembly, but the precise role of the factor remains unclear. Here, we use stable isotope labeling of amino acids in culture and mass spectrometry (SILAC / MS) to examine the function of BipA in ribosome biogenesis. During growth at suboptimal temperature, loss of BipA leads to accumulation of immature large subunit particles (~40S) that lack several proteins. These include L2, L7/12, L10, L14, L16, L17, L19, L27, L28 and L32. Parallel analysis of the control (wild-type) strain shows accumulation of virtually identical intermediate particles, although at much lower levels. Further analysis showed that the main path of 50S assembly differs depending on media in which the cells are grown, demonstrating the robust and flexible nature of the assembly process.

2021-09-09 | PXD017917 | Pride

Overexpressed L20 Rescues 50S Ribosomal Subunit Assembly Defects of bipA-Deletion in Escherichia coli.

Project description:The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.

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Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate.

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