New findings about mechanism of gene regulation is proved through unique crystallization technique

Recently, an article entitled “Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA” was published in the journal of Molecular Cell. The researchers reported the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. By using the innovative crystallization technique, they revealed new insights into the long debated action of the “magic spot”–a molecule that controls gene expression in Escherichia coli and many other bacteria when the bacteria are stressed.

“When bacteria experience stress, such as starvation, they remodel their gene expression,” said Katsuhiko Murakami, professor of biochemistry and molecular biology at Penn State and an author of the paper. In 1969, Cashel and Gallant identified a magic spot (MS) that appeared on a chromatogram made from bacteria that had been starved a key nutrient. Cashel called this molecule, the ‘magic spot,’ because of its appearance from seemingly nowhere when bacteria were starved.

The magic spot then was shown to be guanosine tetraphosphate, or ppGpp, a chemically modified analog of the G nucleotide in the ATCG alphabet of the genome. And it is shown to influence the expression of over 500 genes in response to stress, 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 that produces RNA from genomic DNA. However, still need to study about how this interaction controls gene expression precisely. The new X-ray crystal structures showed three-dimensional images of E. coli RNA polymerase in complex with ppGpp and another important factor that works with ppGpp, DksA for the first time, which provide clues to the interaction.

Even though the three-dimensional structure of RNA polymerase is well established, there are some technical difficulties in seeing the structure of RNA polymerase while it is interacting with other molecules. The interacting molecules often disassociate during the crystallization process necessary to see their structure. By adding molecules of DksA and ppGpp to RNA polymerase that had been crystalized independently, the researchers overcame this difficulty.

“When we soaked RNA polymerase in DksA and ppGpp, we saw that ppGpp bound to the complex of RNA polymerase and DksA in a way that changed the interaction between RNA polymerase and DksA. We think this change could be key to explain how ppGpp alters transcription so that the bacteria can respond to stress.” 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. When ppGpp exists, the expression levels of some genes are turned up, while many are unaffected and some are turned down, that these changed 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.

“We are full of bacteria. They affect our mood, they affect our weight, they affect our immune systems. The ppGpp system is important in lots of these bacteria, allowing them to sense their environment and adjust to stress. Understanding how ppGpp functions will allow us to better understand these bacteria and how they affect us. 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.” said Sarah Ades, associate professor of biochemistry and molecular biology at Penn State and an author of the paper.

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