The Structural and Functional Organization of Ribosomal Compartment in the Cell: A Mystery or a Reality?

Great progress has been made toward solving the atomic structure of the ribosome, which is the main biosynthetic machine in cells, but we still do not have a full picture of exactly how cellular ribosomes function. Based on the analysis of crystallographic and electron microscopy data, we propose a basic model of the structural organization of ribosomes into a compartment. This compartment is regularly formed by arrays of ribosomal tetramers made up of two dimers that are actually facing in opposite directions. The compartment functions as the main 'factory' for the production of cellular proteins. The model is consistent with the existing biochemical and genetic data. We also consider the functional connections of such a compartment with cellular transcription and ribosomal biogenesis.

Application of tyrosine-tryptophan fluorescence resonance energy transfer in monitoring protein size changes.

Monitoring protein size changes has versatile applications in studying protein folding/unfolding, conformational rearrangements, and ligand binding. Traditionally, FRET has been used to obtain this information. However, the use of FRET often requires covalent attachment of exogenous fluorophores. Although intrinsic FRET also exists between tyrosine and tryptophan residues, it has been underused because of tyrosinate formation and spectroscopic overlap. Herein, we clarified the concern of tyrosinate formation and mathematically deconvoluted tyrosine/tryptophan fluorescence spectra. We define a new parameter called FirbY-W (fluorescence intensity ratio between tyrosine and tryptophan) to reflect protein sizes. We demonstrate its applications in studying protein unfolding using several model proteins. In all the cases, our method offers superior sensitivity, data quality, and robustness compared with traditional techniques. The unique power of our method is in its ability to detect elusive conformational changes of intrinsically disordered proteins (IDP). The lack of structure makes IDPs unsuitable for CD or tryptophan fluorescence characterization. Using histone mRNA stem-loop binding protein (SLBP) as an example of disordered proteins, we showed that our method is capable of detecting conformational changes caused by phosphorylation, which are effectively invisible for traditional spectroscopic methods. Our method can also be used to detect RNA binding of disordered proteins.