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.


Bacterial charity work leads to population-wide resistance

Can bacteria employ a population based resistance mechanism to counter drugs?

A study published in Nature, Sept. 2010 suggests this is likely.

Lee et al. show that when a population of E. coli cells was evolved in the presence of norfloxacin, it developed resistance, the level of which was not explained by the resistance of its individual components. That is, this evolved resistant population was composed of a larger proportion of cells that were less resistant to the antibiotic and a smaller proportion that had greater resistance. Their speculation that the high resistant isolates were generating benefits for the low resistant ones led them to find that indole, secreted by the resistant cells was the mediator.

The high resistant isolates had mutations that conferred drug resistance, but were unrelated to indole production. It seems that because these cells are resistant they can produce indole in the presence of antibiotic while the others are inhibited. Furthermore if the gene producing indole is deleted from the high resistant isolate, it can grow better in the presence of antibiotic suggesting that indole production has costs associated with it. But a mixed population with less highly resistant cells and a higher proportion of less resistant cells, could grow in the presence of norfloxacin to substantial levels, only if the highly resistant cells were capable of producing indole. Indole seems to enable antibiotic detoxification in the less resistant isolates by up-regulating export pathways and oxidative stress protective mechanisms.

Hence, the altruistic production of indole by the high resistant isolate enables the population as a whole to grow in the presence of otherwise inhibitory concentration of norfloxacin.

The authors suggest that:

This altruism allows weaker constituents to survive and concurrently explore the space of beneficial mutations, a phenomenon similar in character to kin selection. These few drug-resistant mutants, by enhancing the survival capacity of the overall population in stressful environments, may also help to preserve the potential for the population to return to its genetic origins should the stress prove transient.

What’s more, probing into ways in which a population reacts to the presence of antibacterial substances to gain resistance will help develop means to intervene this phenomenon.

Translation-dependent localisation of DNA loci to the membrane

The process of transertion, in which co-transcription and translation of membrane proteins could occur concomitantly with the insertion of the protein into the membrane, had been hypothesised, but never been proven (to our knowledge). In a recent paper published in PNAS, Libby and colleagues, show just that by using E. coli as the model system.

They interpret their findings in the context of its possible impact on DNA conformation:

membrane protein expression across the entire genome is likely to play a key role in shaping chromosome conformation. Our results further suggest that repositioning at any given locus is likely to be transient, occurring concomitantly with bursts of transcription. The resulting movement toward and away from the membrane at points distributed around the chromosome may be an important mechanism for maintaining the nucleoid in a sufficiently dynamic state to ensure accessibility to regulatory proteins, ribosomes, and RNA polymerase

Will this feed back to transcription? Can this feature, assuming that it is strong enough a selective force, influence gene organisation?