Mechanisms of RNA processing and decay that are dependent on RNaseE and related enzymes

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In response to changing and often adverse or competitive environments, organisms fine-tune their metabolism and composition via changes in the expression of genes (the basic units of information inherited in all organisms) in order to maximize survival and growth. An important model for studying gene expression is the gut-dwelling bacterium E. coli. This is due in part to the ease with which this organism can be manipulated genetically and grown using simple media. It was experiments with E. coli that finally proved that DNA encodes the inheritable information (Hershey & Chase), deciphered the relationship between the information in genes and the amino acid building blocks of proteins (Nirenberg & Khorana), which form most of the working parts of cells, and demonstrated that E. coli can sense the availability of nutrients and then adjust its metabolism to use first those that provide the most 'energy' (Jacob & Monod). It was found, as part of work on the latter, that the synthesis of proteins required for the utilisation of a sugar was terminated rapidly when the sugar was no longer available. This led to the suggestion, which has now been proved, that protein is encoded via an unstable messenger that rapidly disappears when its synthesis is blocked. This intermediate is now known to be composed of RNA and is in effect a 'copy' of the information in genes. The process of making messenger RNA from DNA (transcription) and the process of making proteins from mRNA (translation) have been studied extensively using biochemistry and genetics. More recently, the solving of atomic-resolution structures of the machines that mediate transcription and translation has provided paths to an understanding of the molecular mechanisms at the heart of these steps in gene expression. Although the synthesis of mRNA and its subsequent translation are clearly important steps, the rate of decay of any RNA is just as important as its rate of synthesis in determining the cellular levels. Consequently, the stability of mRNA is a key determinant of the amount of protein produced from a gene. Despite the central importance of mRNA decay, our understanding of this process lags behind that of transcription and translation. Excellent progress is however being made. A major breakthrough in the study of mRNA decay in E. coli was the identification of an essential gene that is required for the normal rapid decay of many mRNAs. This gene has been shown not only to encode an endoribonucleolytic activity (RNaseE), but also to serve as a platform for the assembly of a machine called the degradosome, which contains other enzymes important for rapid mRNA decay. To better understand the process of mRNA decay in E. coli, we have investigated the factors that control the cleavage of RNA by RNaseE. This has involved using chemistry to synthesize substrates that can be used in biochemical assays and genetics to knockout or modify gene function in vivo. Biophysical techniques have been used to establish the overall structure of the catalytic domain of RNaseE and features that are required for both its assembly and ribonucleolytic activity. Most recently, one of our collaborations has led to the solving of the crystal structure of the catalytic domain of E. coli RNaseE. Using the considerable detail this has provided at the atomic level, our overall objective now is to establish the contribution of specific molecular traits of RNaseE (and associated proteins) to the pattern of RNA processing and decay observed in E. coli. This in turn may permit more efficient use of E. coli as a host for producing biomolecules of commercial or medical importance and could eventually be useful in the development of antibacterial drugs that target mRNA decay mechanisms.