E. coli RNA helicase DbpA and its role in assembly of the large subunit of the ribosome

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Assembly of the large and small subunits of the <em>E. coli</em> ribosome is a complicated process that involves transcription, processing, and modifying of the ribosomal RNA, the binding of ribosomal proteins, and the folding of the particles into complex structures. A number of ribosome assembly factors, including maturation factors, GTPases, and DEAD-box proteins, have been recently identified to associate transiently with the growing ribosome and assist assembly. Understanding the molecular mechanisms of these factors is important for understanding how ribosomes are made. E. coli</em> DEAD-box protein A (DbpA) is an RNA-stimulated ATPase and helicase that has been implicated in the assembly of the 50S subunit of the ribosome. Although biochemically well-characterized, the molecular function was still undefined. Therefore, in order to identify a phenotype for DbpA, eleven single-point mutations in the conserved ATPase motifs (I, II, III, and VI) were designed to disrupt function of DbpA without affecting binding to its RNA substrate. <em>In vitro</em> biochemical assays of the purified mutant proteins revealed significantly reduced ATPase and helicase activities but wild type affinities for the 23S rRNA substrate. Interestingly, only one of the eleven mutants, R331A, resulted in a slow growth phenotype that was found to be due to a decrease in functional 70S ribosomes and accumulation of an inactive 45S particle. Analysis of this 45S particle revealed that it contains incompletely processed and undermodified 23S rRNA and reduced levels of five late assembly L-proteins. These five L-proteins are located in the same region that DbpA binds to, which is consistent with the absence of a conformational rearrangement necessary for their binding at this site. In addition, 45S particles could stimulate DbpA to hydrolyze ATP nearly as well as 23S rRNA, suggesting that the DbpA binding site in the particles is accessible in contrast to mature 50S subunits, which are not able to stimulate DbpA activity. Therefore, the 45S particles accumulate due to the inactivity of the R331A mutant, although the exact stage that this occurs is still unknown. In summary, these data support the hypothesis that DbpA participates in a conformational rearrangement necessary for maturation of the 50S subunit.

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  • 09/19/2018
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