Research

General Research Procedure in Ke Lab

CRISPR interference in Prokaryotes

The use of small RNAs to regulate gene expression is ubiquitous in all living organisms including bacteria. In one remarkable instance, bacteria and archaea acquire resistance to invading foreign nucleic acids - such as conjugative plasmids, transposable elements and phages - by employing an RNA-mediated defense mechanism. In this process, short fragments (~24 to 48 nucleotides) of the invading DNA are integrated in the genome as spacers between similarly sized clusters of regularly interspaced short palindromic repeats (CRISPRs). CRISPRs are a novel class of repetitive DNA that have been identified in 88% of the archaeal genomes and 39% of the bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter jejuni, Clostridium botulinum, Listeria monocytogenes, Mycobacterium tuberculosis, Yersinia pestis, and enteropathogenic and enterohaemorrhagic Escherichia coli. Adjacent to the CRISPR repeats and spacers is a set of conserved CRISPR-associated (cas) genes that encode the Cas proteins. Owing to its widespread occurrence, the CRISPR defense system has attracted a great deal of attention.

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 S_MK and the B. subtilis S-box.