Structural insights into the assembly of the 30S ribosomal subunit in vivo： functional role of S5 and location of the 17S rRNA precursor sequence

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coil strain （ArsgAArbfA）. The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, inter- mediates derived under the two contrasting salt condi- tions （high and low） likely reflect two distinctive assembly stages, the relatively early and late stages of the 3＇ domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5＇ end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal thelocation of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mech- anisms on subunit production and protein translation.

Identifi cation and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1

Arabidopsis BOTRYTIS-INDUCED KINASE1(BIK1)is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity.It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-asso-ciated molecular patterns(PAMPs).Although site-specifi c phosphorylation is speculated to mediate the activation and function of BIK1,few studies have been devoted to complete profiling of BIK1 phosphorylation residues.Here,we identified nineteen in vitro autophosphoryla-tion sites of BIK1 including three phosphotyrosine sites,thereby proving BIK1 is a dual-specifi city kinase for the fi rst time.The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spec-trometry(MS).Thr-237,Thr-242 and Tyr-250 were found to most signifi cantly affect BIK1 activity in autophosphoryla-tion and phosphorylation of BAK1 in vitro.A structural model of BIK1 was built to further illustrate the molecular functions of specifi c phosphorylation residues.We also mapped new sites of FLS2 phosphorylation by BIK1,which are different from those by BAK1.These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and medi-ates downstream signaling specifi city.