Hfq, a protein with RNA chaperone activity ?

Research project P 14486 Hfq, a protein with RNA chaperone activity ? Udo BLÄSI 26.6.2000 Besides its function in RNA phage Qß replication, Escherichia coli host factor I (Hfq /HF1) has been suggested to bind to a number of different mRNAs which appears to results in translational activation or repression. We have recently shown that Hfq binds to the 5`UTR of the E. coli ompA mRNA and prevents ribosome binding. In contrast, in S. typhimurium Hfq seems to act as a translational activator of the rpoS gene encoding the stationary phase s factor which in turn is a regulator for the expression of virulence genes. The aim of this study is twofold: We will attempt to solve the solution structure of Hfq by NMR analysis and decipher surface-exposed amino-acid residues required for RNA binding. A targeted mutational analysis will then be performed to verify the structure- function predictions. Several approaches will be taken to study the biochemistry of the protein and its interaction with RNA. The sequence and structural requirements of RNA for Hfq binding will be evaluated by footprinting, chemical probing and chemical interference experiments. There is some evidence that Hfq acts as an RNA chaperone, a steadily increasing class of proteins which appear to promote structural rearrangements in RNAs. The interaction of RNA chaperones with their substrate is poorly understood. Using a novel functional in vivo assay as well as an in vitro assay, we will determine whether Hfq possesses RNA chaperone activity. A proteome analysis upon depletion for and overproduction of Hfq will be carried out. It is anticipated that this study will reveal target genes which are under positive as well as under negative control of Hfq. It will shed light on the physiological role of Hfq and should uncover whether Hfq is required for the regulation of a subset of genes. In addition, the sequence and structural analysis of the Hfq-target mRNAs identified with the proteome approach has the potential to reveal a RNA binding motif(s) for Hfq. It is anticipated that the combination of biochemical and genetic approaches will shed new light on the interaction of a (putative) RNA chaperone with its substrate.