Molecule That 'Blocks' Key Bacterial Enzyme May Lead To New Antibiotics

Two teams of researchers at Rutgers, The State University of New Jersey, discovered independently that MccJ25 uniquely blocks a "tunnel" into the bacterial enzyme, RNA polymerase (RNAP). The "tunnel" is used to bring raw materials for RNA synthesis into the enzyme and to expel byproducts of RNA synthesis.

"Closing the crowded, two-way 'tunnel' starves RNAP, shuts it down and kills the bacteria," said Richard H. Ebright, a Howard Hughes Medical Institute investigator, and a professor in Rutgers' department of chemistry and chemical biology and the Waksman Institute of Microbiology. "Understanding the way in which MccJ25 works sets the stage for the development of novel antibacterial drug designs."

To understand how MccJ25 works, Ebright's group used genetic methods to test hundreds of thousands of RNAP derivatives, or variants, in order to define the binding sites for MccJ25 on RNAP. The researchers also used biophysical methods, attaching fluorescent tags to MccJ25 and to each of a dozen sites in RNAP. Using the tags, the researchers gauged the position of each bound pair in a GPS-like manner, verifying the results of the genetic work. They then used biochemical methods to find out what happened once MccJ25 binds to the RNAP.

The research team of Konstantin Severinov, an associate professor in Rutgers' department of molecular biology and biochemistry and the Waksman Institute, had been the first to demonstrate that RNAP from cells resistant to MccJ25 also showed resistance to the drug in a test tube. In their current work, these researchers used biochemical methods to characterize, in molecular detail, the mechanism of MccJ25 action. In addition, the group used sophisticated biophysical methods that revealed how MccJ25 binds to a single RNAP molecule, stopping it instantaneously.

In the June 18 issue of the journal Molecular Cell, Ebright and colleagues, and Severinov and members of his research team, describe in separate reports how each team used different experimental methods to reach the same conclusions.

"It was only last year that we solved the structure of this molecule, just 21 amino acids long, remarkable both in its size and its structure," said Ebright, a participant in the original structural studies, along with Severinov.

"A great many papers have been published on this molecule in the last year," added Severinov. "Interest in MccJ25 has really exploded."

Shaped like a lasso with its tail pulled back through the loop, MccJ25 is one of very few molecules known to have this highly stable and rigid configuration – a characteristic that may lead to applications beyond drug design.

"MccJ25's sturdy structure permits it to withstand all sorts of harsh environmental conditions," said Severinov. Because of its robustness, he added, "the [U.S.] Department of Defense is interested in the molecule as a potential bacterial decontaminating agent."

MccJ25 inhibits bacterial RNAP and kills bacteria, but it does not inhibit human RNAP and would therefore not kill a human. Ebright cautions, however, that MccJ25 has some shortcomings. It affects only E. coli and closely related bacterial species and is highly subject to developing resistance.

Rutgers researchers are already at work on overcoming these obstacles. Ebright's group has modified the molecule to produce derivatives that have a narrower resistance spectrum. The Severinov group is pursuing the same goal through a National Institutes of Health joint research program with scientists from the University of Tucuman, Argentina, who originally discovered MccJ25 in 1992.