Hfq variant with altered RNA binding functions.
The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS-lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq-RNA interaction but has probably no effect on the folding and the oligomerization of the protein.
Ziolkowska
Katarzyna
K
Régulation de l'Expression Génétique chez les Microorganismes, UPR CNRS no. 9073 conventionnée avec l'Université Paris 7, Denis Diderot, Paris, France.
Derreumaux
Philippe
P
Folichon
Marc
M
Pellegrini
Olivier
O
Régnier
Philippe
P
Boni
Irina V
IV
Hajnsdorf
Eliane
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D035001:Host Factor 1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D011062:Polynucleotide Adenylyltransferase