Poster abstracts

Poster number 16 submitted by Priyanka Das

Ribosome binding domain of L13a: Relationship with ribosome release and phosphorylation

Priyanka Das (Dept of Biological, Geological and Environmental Sciences, Cleveland State University), Purvi Kapasi (BGES, Cleveland State University), Sujan Chaudhuri (BGES, Cleveland State University), Keyur Vyas (BGES, Cleveland State University), Anton A. Komar (BGES, Cleveland State University), Barsanjit Mazumder (BGES, Cleveland State University)

Abstract:
We have shown that the release of ribosomal protein L13a from 60S ribosome and its subsequent phosphorylation is a key step in the formation of GAIT complex in transcript specific translational silencing. In order to gain insight into the mechanism of release, it is necessary to determine the domain of L13a responsible for binding to 60S ribosome. Using the ribosome incorporation assay of the ectopically expressed tagged L13a we have identified ribosome-incorporation defective mutants of L13a. We have performed structural homology modeling of human L13a based on the crystal structure of prokaryotic L13 (the homolog of mammalian L13a), which identified a loop in L13a, harboring Arg at position 68 at the tip of the loop. in silico analysis of L13a has predicted the amino acids most likely to contact RNA in solved complex structure from protein data bank. This analysis also identified several candidate amino acid residues and motif that potentially bind to rRNA. In consistence with the above two models, we have experimentally verified Arg 68 and triple mutant Arg-Lys-Arg, 59-60-61 as ribosome incorporation defective L13a. We then tested the role of other predicted amino acid residues, which showed no incorporation defect. We will confirm the incorporation defective mutants by testing the in vitro direct binding between purified 60S ribosome and recombinant His tagged L13a using sucrose gradient analysis. In consistence with the ribosome incorporation assay our recent result using immunoprecipitation coupled with RT-PCR shows direct binding between wild-type L13a, however Arg 68 mutant failed to bind. Imaging studies show no defect in the nuclear translocation step of the mutant. Interestingly no difference was observed in the IFN-γ mediated delayed phosphorylation between the wild-type or incorporation defective mutant.

Keywords: L13a, Incorporation Defect, Direct Binding