The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion

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.

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Making a RNA polymerase out of a DNA polymerase

DNA polymerases build DNA from dNTPs and enable replication, whereas RNA polymerases make RNA from NTPs, permitting transcription. Though the subunit structures of DNA and RNA polymerases appear different, there are several single-subunit RNA polymerases in mitochondria and T-odd bacteriophages, which are thought to have evolved from an ancestral DNA polymerase. Now the question that arises is what are the changes that can make a DNA polymerase into an RNA polymerase; should be a difficult change to make given that in the presence of a vast excess of NTPs over dNTPs, DNA polymerases have to powerfully discriminate between the two substrates.

Though several mutations have been described so far, which allow a DNA polymerase to bind to NTPs and produce short stretches of RNA (typically <10nt) at which point the mutant polymerase simply stalls, the answer to the question above has remained elusive.

In a recent paper published in PNAS, Cozens and colleagues from the MRC-LMB and the NIH demonstrate that two targeted mutations in the replicative polymerase from Thermococcus gorgonarius can make an RNA polymerase out of it. The determining mutation is located at about 25 Angstroms from the active site in what is called a ‘thumb’ subdomain. The combination of the two mutations makes the DNA polymerase synthesise RNA upto 1.7kb long, and use various unnatural nucleotide substrates as well. The mutation hardly affects any of the enzyme kinetic parameters associated with its natural dNTP substrate. However, for NTPs (the RNA substrate), the mutation pair increases the Vmax 4-fold and decreases the Km (thus increasing substrate affinity) by three orders of magnitude. These effects might be explained by steric effects and charge differences in the thumb subdomain between the two forms of the polymerase.

The residue at the determining position in the thumb subdomain, though conserved in related organisms, varies in distant organisms. On the basis of the high-temperature adaptation of Thermococcus, the authors state that this residue (referred to as a ‘steric gate’)

may therefore be a specific adaptation to prevent the deleterious consequences of NTP mis-incorporation or replication of genomic lesions at high temperature. Conversely,…, in analogy  to the diverse nature of steric-gate residues, the second gate may therefore be elaborated in different ways in the context of different thumb domain structures.