ABSTRACT
We have proposed that the ancient ribosome increased in size during early evolution by addition of small folding-competent RNAs. In this Accretion Model, small RNAs and peptides were subsumed onto subunit surfaces, gradually encasing and freezing previously acquired components. The model predicts that appropriate rRNA fragments have inherited local autonomy of folding and local autonomy of assembly with ribosomal proteins (rProteins), and that the rProtein and rRNA are co-chaperones. To test these predictions, we investigate the rRNA interactions of rProtein uL23 and its tail, uL23tail, which is a ß-hairpin that penetrates deep into the core of the large ribosomal subunit. In the assembled ribosome, uL23tail associates with Domain III of the rRNA and a subdomain called "DIIIcore". Here using band shift assays, fluorescence Job plots, and yeast three-hybrid assays, we investigate the interactions of rProtein uL23 and its tail with Domain III and with DIIIcore rRNA. We observe rRNA1-uL23tail1 complexes in the absence of Mg2+ ions and rRNA1-uL23tailn (n > 1) complexes in the presence of Mg2+ ions. By contrast, the intact uL23 rProtein binds in slightly anticooperative complexes of various stoichiometries. The globular and tail regions of rProtein uL23 are distinctive in their folding behaviors and the ion dependences of their association with rRNA. For the globular region of the rProtein, folding is independent of rRNA, and rRNA association is predominantly by nonelectrostatic mechanisms. For the tail region of the protein, folding requires rRNA, and association is predominantly by electrostatic mechanisms. We believe these protein capabilities could have roots in ancient evolution and could be mechanistically important in co-chaperoning the assembly of the ribosome.
Subject(s)
Evolution, Molecular , Models, Molecular , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Cations, Divalent/metabolism , Electrophoretic Mobility Shift Assay , Escherichia coli , Hydrogen Bonding , Magnesium/metabolism , Protein Binding , Protein Domains , Protein Folding , Protein Structure, Secondary , RNA, Bacterial/metabolism , Thermus thermophilus , Two-Hybrid System TechniquesABSTRACT
Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates â¼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
Subject(s)
Evolution, Molecular , Models, Genetic , Ribosomes/genetics , Magnesium/chemistry , Models, Molecular , Nucleic Acid Conformation , Peptide Fragments/chemistry , Protein Binding , RNA Cleavage , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/genetics , RNA, Ribosomal, 23S/metabolism , Ribonuclease H/chemistry , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Thermus thermophilus/geneticsABSTRACT
Diapausing embryos (resting eggs) from brachionid rotifers are able to withstand desiccation and thermal stress. Resting eggs can remain viable for decades, and develop normally once placed in a permissive environment that allows for hatching, growth and development. The exact mechanisms of resistance are not known, although several molecules have been suggested to confer protection during desiccation and thermal stress. In this study, we have identified by mass spectrometry two thermostable proteins, LEA (late embryogenesis abundant) and VTG (vitellogenin-like), found exclusively in the resting eggs of Brachionus manjavacas. This is the first observation that LEA proteins may play a role in thermostability and the first report of a VTG-like protein in the phylum Rotifera. These proteins exhibited increased expression in rotifer resting eggs when compared to amictic females. Our data suggest the existence of alternate pathways of desiccation and thermal resistance in brachionid rotifers.
