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General Research Procedure in Ke Lab




Significant Studies in Ke Lab

Exosome

In eukaryotic cells, stable RNAs are transcribed with a 3’ extension that is subsequently trimmed from 3’-to-5’, whereas aberrantly processed RNAs are subject to rapid degradation. A conserved 300 – 400 kDa exoribonuclease protein complex, the exosome, plays crucial roles in both the RNA 3’-end formation and turnover processes. The nuclear exosome is required for the 3’ end formation of 5.8S rRNA, small nuclear and nucleolar RNAs; and involved in the degradation of inefficiently spliced and hyper– or hypo-adenylated pre-mRNAs. The cytoplasmic exosome is required for the degradation of normal mRNAs as well as those containing premature termination codons, lacking termination codons, or bearing AU-rich elements near the 3’ untranslated region. Using X-ray crystallography and biochemical tools, we aim to understand the architecture, working mechanism, and the regulation of this multi-subunit machinery.


Riboswitch

Riboswitches are structured RNAs that recognize specific small molecules, usually key metabolites inside the cell, and “switch” on or off gene expression at either transcription or translation level. The discovery of these short cis-acting RNA elements has drastically changed our understanding of gene regulation. Majority of riboswitches were found in prokaryotic genomes, while only a few examples of eukaryotic riboswitches have been reported. Riboswitches are especially prevalent in Gram-positive bacteria, exemplified by Bacillus subtilis as a model organism, but also in a number of important pathogens such as Bacillus anthracis, Staphylococcus, Enterococcus, Streptococcus, Listeria, Clostridium, and Mycobacterium. This and other characteristics attract increasing attention to target riboswitches for antibiotic development.

Of the twelve different riboswitch classes that recognize nine different key metabolites, S-adenosyl methionine (SAM) riboswitches are the most commonly found. For example, nearly half of all known riboswitches identified in B. anthracis bear consensus primary sequence and secondary structures of the S box motif. Three classes of SAM riboswitches have been found. They contain completely different secondary structures, representing nature’s three independent RNA solutions to achieve specific SAM recognition. We recently determined the crystal structures of two (out of three) SAM riboswitch classes: the E. faecalis SMK and the B. subtilis S-box.
 

Bacteriophage Phi29 Packaging RNA

How bacteriophage f29 packages its genome into protein capsids is a mystery and a marvel. The DNA genome is packed to near crystalline density inside the capsid, against internal pressure of up to 2000 psi, through a molecular motor composed of a dodecameric head-tail protein connector, and a pentameric prohead RNA (pRNA) and ATPase gp16. We are working on the structures of the packaging motor components to explain how this natural nano-machine works and why RNA is needed as a component.



Signal Recognition Particle (SRP)
RNA-protein (ribonucleoprotein) complexes, such as the ribosome, signal recognition particle (SRP), spliceosome, and telomerase, carry out essential functions inside cells. We are currently investigating the structural mechanism of SRP-mediated co-translational translocation of proteins across or into cell membranes. In this vital cellular process, SRP recognizes the hydrophobic signal sequence of the nascent polypeptide emerging from the ribosome, resulting in transient elongation arrest in eukaryotes, and targets the ribosome to the membrane via a GTP-dependent interaction with the SRP receptor (SR).