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

Reference	BB/D016096/1
Principal Investigator / Supervisor	Dr Kenneth McDowall
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Inst of Integrative & Comparative Biolog
Funding type	Research
Value (£)	278,327
Status	Completed
Type	Research Grant
Start date	01/11/2006
End date	31/10/2009
Duration	36 months

Abstract

With the move towards more quantitative and predictive biology, any models for gene regulation will be unbalanced unless understanding improves of RNA decay and processing. E. coli is an excellent model: it is manipulated readily and many of its regulatory components are conserved in most, if not all branches of life. The decay of RNA in E. coli is rapid and 'all-or-none' in nature, and is frequently controlled by 5'-end determinants. Many, if not most transcripts are decayed by mechanisms that are dependent on RNaseE. This endonuclease is also involved in the processing of rRNA, which it cuts at only a few sites despite having little sequence specificity. RNaseE is associated with other mRNA-decay factors in the degradosome complex and a 5' -monophosphate group on RNA stimulates its activity. While the central importance of RNaseE is established, there is as yet no overall understanding of the contribution of its various domains to the patterns of RNA processing and decay observed in vivo. As part of previous funding, we created mutants that are not stimulated by a 5' monophosphate group. We propose now to continue our analysis of RNaseE with the specific aims of determining whether stimulation by a 5'-monophosphate group is (i) critical for the ordered and/or limited cleavage of rRNA, (ii) endows core characteristics of mRNA decay, i.e. its 'all-or-none' nature and 5' to 3' directionality, and (iii) contributes to initial cleavage should a 'decapping' mechanism exist. These aims will be addressed using 5'-sensor mutants. We will also extend our studies to include domains required for the fastest rates of decay, and growth competitiveness. The specific aims in this area are to test whether (iv) a RNA-binding domain outwith the catalytic domain facilitates cleavage of sub-optimal sequences, and (v) has a role in dictating where cleavage occurs. We will also (vi) investigate the mechanism by which an RNA helicase in the degradosome assists RNaseE.

Summary

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.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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