Identification Of LepA As Ppiase In Translation Regulation

During translation, the ribosome moves along mRNA in a one-waydirection--from 5'to 3'--and converts the genetic information into a polypeptide chain.Recently, with the finding and identification of the highly conserved protein LepA,this directional movement turned out to be incomplete, a new kind of tRNAmovement inside the ribosome, namely the back-transloation of the tRNAs andmRNA in the 3'to 5'direction by one codon distance during the translation elongationcycle is discovered. LepA is a highly conserved protein in bacteria and bacteriumderived organelles in eukaryotes. Sequence and structural data show that it isconstituted of five structural domains, in which domains I to IV are homologous todomains I, II, III, and V of elongation factor G (EF-G). LepA does not have regionscorresponding to EF-G domains G'or IV but has a unique C-terminal domain(LepA-CTD) with specific motif not found on any other protein. The lack of domainIV on LepA allows tRNA to occupy the A site simultaneously with LepA,directinteractions between LepA and tRNA and the strong positive charge in the C-terminaldomain of LepA bring it a reverse translocase activity. By doing so, LepA makes thetranslocation more flexible that might be of great importance for the protein synthesisunder special physiological conditions.Cryo-EM reconstruction demonstrated that LepA interacts with the ribosome inthe same orientation as EF-G and catalyzes GTP hydrolysis in the classicaltranslational GTPase manner through the"GTPase-associated center"(GAC), whichis responsible for binding and stimulation of the GTPase activity of translationalGTPase throughout the translation process. Three GAC components are considered tobe important: the sarcin-ricin loop (SRL) in helix 95 of the 23S rRNA, the ribosomalL7/L12 stalk and the stalk base(SB)- L11 and its binding site on H43 and H44 of the23S rRNA.Here we identify a new enzymatic activity of LepA as peptidyl-prolyl cis-transisomerase (PPIase) and locate the PPIase center on LepA's G-domain and domain V.The physiological substrate of the PPIase is targeted at the ribosomal protein L11 inSB by chemical cross-linking and NMR analysis. L11 is a high flexible component, consisting of two domains: a compact CTD responsible for the tight binding with H43and H44, a loosely folded N-terminal domain (NTD) with high freedom and a shortlinker connecting them. By interacting with the conserved proline residues onL11-NTD, LepA can accelerate their cis-trans isomerization dramatically so to initiatethe downstream conformational change which is important for connecting L7/L12.Protein L7/L12 plays an important role in activating the GTP hydrolysis andcontrolling the Pi release. More importantly, PPIase activity is also found on the otheressential translational GTPase. That implies a general role of translational GTPase inmodulating the ribosome through proline switch to function coordinately andefficiently.