Many species of bacteria – including those that infect humans – are themselves threatened by parasites and predators. A large group of viruses called bacteriophages are known to infect bacteria. On infection, the phage transfers its DNA to the bacterial cell, hijacks the bacterial DNA-replicating machinery to make multiple copies of itself, and then escapes by killing the bacterial cell.
Bacteria have in turn evolved a variety of immune mechanisms to protect themselves against invading phages. About 40% of sequenced bacterial genomes contain a set of CRISPR/Cas genes. These include a set of genes encoding Cas proteins as well as CRISPR loci, which are arrays of short repeats separated by highly variable ‘spacer’ sequences.
These spacer sequences are identical to sequences present in phage DNA. They are transcribed into small RNA molecules called crRNAs, which, with the help of the Cas proteins, bind to and cause degradation of the invading phage DNA. However, a recent study published in Nature has discovered a novel CRISPR/Cas system – not in a bacterium, but in a bacteriophage.
This bacteriophage, called ICP1, attacks a strain of the cholera pathogen Vibrio cholerae. The authors demonstrated that the CRISPR/Cas system in the bacteriophage is fully functional and targets a region of bacterial DNA that is responsible for defense against the phage. Not only is this an example of a supposedly bacterial immune mechanism being used by a phage, but it appears that the phage uses it to counter an entirely different bacterial immune mechanism. This host vs. pathogen arms race suddenly looks a lot more interesting.
The authors of this recently published paper in nature structural & molecular biology (http://www.nature.com/nsmb/journal/v20/n2/full/nsmb.2472.html) provide many arguments against contribution of facilitated diffusion (1D hopping/sliding along the DNA) as a promoter search mechanism for Escherichia coli RNA polymerase. According to them the contribution of 3D diffusion, especially at physiological protein concentrations outweighs the contribution of any form of facilitated diffusion.
Their experimental system involves a curtain of λ dna molecules tethered at both ends in the same orientation. Using quantum dot tagged RNAP they were able to visualize the RNAP molecules at the DNA curtain using TIRFM. Based on the lifetimes of the quantum dot labeled single molecules of RNAP they discriminate various intermediates: (in order of increasing lifetimes) random diffusion in absence of DNA interaction, random interactions with DNA, closed complexes and open complexes. They find that most events where RNAP engages the promoter were preceded by 3D diffusion and 1D diffusion was virtually not seen.
They also come up with a theoretical model to determine the significance of contribution of the various forms of diffusion to promoter search. They find that with greater concentrations of the protein, 3D diffusion overcomes any possible accelerating effects of 1D diffusion and thus come up with the concept of ‘facilitation threshold’, the concentration of (any) DNA-interacting protein below which facilitated diffusion would be faster in target search than 3D diffusion. They surmise that for the levels of RNAP in the cell 3D diffusion would be a faster mechanism for promoter search.
To demonstrate the significance of facilitation threshold experimentally they use the lac repressor and insert tandem lac operator sequences in the λ DNA curtain. Under conditions where non-specific DNA binding and hence facilitated diffusion is favoured they see that the lac repressor at low concentrations engages its operator mainly by 1D diffusion, however when the concentration of the repressor was increased there was an increase in the number of events in which operator binding was preceded with 3D diffusion of repressor rather than 1D diffusion clearly adding weight to the concept.
Finally the authors also discuss how under various in-vivo conditions seen by the RNAP like presence of nucleoid associated proteins and higher chromatin architecture as well as molecular crowding why 3D diffusion would be a more prevalent mechanism for promoter search rather than 1D diffusion.