Structural And Functional Study Of A Conserved GTPase YihA In Prokaryotic Ribosome Biogenesis

Ribosome-catalyzed protein biosynthesis is a fundamental process for allcellular activities. As a consequence, the assembly of the ribosome itself isessential for the cell growth and regulated by various growth-control pathways.A number of ribosome-dependent GTPases, with proposed functions in ribosomebiogenesis, are believed to be key components in cell cycle regulation. Forexample, YihA, an essential GTPase widespread in bacteria is required for the50S subunit biogenesis and is also involved in cell cycle control. With acombination of cryo-electron microscopy reconstruction and biochemicalapproaches, we explore the molecular mechanism and structural basis of thefunctional role of YihA in the assembly of the50S ribosomal subunit in E. coli.Our results show that YihA has a strong interaction with the50S subunit invitro, and this association can be enhanced by guanine nucleotides, with itsaffinity to the50S subunit increasing, in an order of GTP, GDP and GMPPNP.Following this clue, we determined cryo-EM structures of two YihA-boundstates of the50S subunit,50S-YihA-GMPPNP and50S-YihA-GDP complexes.Our structural data show that YihA binds to the exposed side of H69of the23SrRNA, which is an important intersubunit bridge for the association of the50Sand30S subunits to form70S ribosomes, and leads to dramatic conformationalchanges on the23S rRNA, especially at regions of H68-71. These data suggestthat the binding of YihA to the mature50S subunit might have reversed the50Ssubunit to a conformational state similar to late-stage50S assemblyintermediates.Guided by our structural information, we further tested the binding of YihAto different segments of the23S rRNA. As a result, YihA has a strong affinityfor in vitro transcribed H68-71segment of the23S rRNA, which is consistentwith our cryo-EM data. Through the analysis of YihA gene-specific transposoninsertion mutants, we found that dysfunction of YihA causes cellular defects inthe50S subunit maturation, with an accumulation of premature50S particles inthe cell. The sedimentation coefficient of the premature50S particles is approximately45S, and it shows lack of several ribosomal proteins and a fewextra proteins compared to the mature50S subunit.To further explore the functional roles of the N-terminal and the C-terminalsequences of YihA, we built YihA truncated mutants. In vitro binding assaysand cryo-EM structure both show that both the N-terminus (15amino acids) andthe highly charged C-terminus (30amino acids) are important for the function ofYihA.Together with published data, we propose a possible molecular mechanism inwhich YihA acts as a molecular chaperone on RNA folding in the late stage of the50Ssubunit maturation. Particularly, YihA plays an important role in the assembly ofH68-71. The folding of the long helix of H68-71is probably a critical rate-limiting stepduring the late-stage biogenesis. In addition, at this strategic location, YihA likelyfunctions as a checkpoint protein to prevent immature50S particles into translationcycle.