Using an innovative crystallization technique for studying 3D structures of gene transcription machinery, researchers discovered new insights into the long debated action of the 'magic spot' a molecule that controls gene expression in E. coli and many other bacteria when the bacteria are stressed. The study has published in the journal Molecular Cell.
The study contributes to fundamental understanding of how bacteria adapt and survive under adverse conditions and provides clues about key processes that could be targeted in the search for new antibiotics. The magic spot subsequently was shown to be guanosine tetraphosphate, or ppGpp, a chemically modified analog of the G nucleotide in the ATCG alphabet of the genome.
Its appearance following starvation and other stresses is associated with changes in the expression of over 500 genes, most prominently genes for the structural RNAs that are components of the ribosome the enzyme responsible for protein synthesis. The ppGpp molecule interacts with E. coli's RNA polymerase the cellular machine that produces RNA from genomic DNA but precisely how this interaction controls gene expression remains a mystery.
The new X-ray crystal structures, however, provide clues to this process by showing for the first time three-dimensional images of E. coli RNA polymerase in complex with ppGpp and another important factor that works with ppGpp, DksA.
The three-dimensional structure of RNA polymerase is well established, but seeing the structure of RNA polymerase while it is interacting with other molecules has proved to be technically difficult. The interacting molecules often disassociate during the crystallization process necessary to see their structure. The researchers overcame this difficulty by adding molecules of DksA and ppGpp to RNA polymerase that had been crystalized independently.
"We first created crystals of RNA polymerase, then soaked in DksA and ppGpp," said Vadim Molodtsov, assistant research professor in biochemistry and molecular biology at Penn State and another author of the paper. RNA polymerase in bacteria controls the expression of all genes, but in response to the presence of ppGpp, the expression levels of some genes are turned down, while many are unaffected and some are turned up.
These changes in expression levels allow the bacteria to alter their composition to better survive stress. The researchers speculate that the different responses may be due to individual differences in the promotors. DNA sequences near the beginnings of genes that initiate expression of individual genes.
The ppGpp system is important in lots of these bacteria, allowing them to sense their environment and adjust to stress. The system is also important in bacterial pathogens that cause infectious disease. Understanding how ppGpp works could allow us to find ways to disrupt its functions and develop new antibiotics.