TY - JOUR
T1 - Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing
AU - Panja, Subrata
AU - Santiago-Frangos, Andrew
AU - Schu, Daniel J.
AU - Gottesman, Susan
AU - Woodson, Sarah A.
N1 - Funding Information:
This work was supported by a grant from the National Institute of General Medicine ( R01 GM46686 to S.W.) and in part by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research (to S.G.)
Funding Information:
This work was supported by a grant from the National Institute of General Medicine (R01 GM46686 to S.W.) and in part by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research (to S.G.). The authors thank N. Majdalani and Y. Peng for technical help, as well as N. De Lay, G. Storz, T. Updegrove and A. Zhang for discussion and comments.
Publisher Copyright:
© 2015 Elsevier Ltd.
PY - 2015/5/25
Y1 - 2015/5/25
N2 - Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.
AB - Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.
KW - Hfq
KW - RNA chaperone
KW - RNA protein interactions
KW - molecular beacon
KW - small non-coding RNA
UR - http://www.scopus.com/inward/record.url?scp=84952863347&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84952863347&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2015.07.010
DO - 10.1016/j.jmb.2015.07.010
M3 - Article
C2 - 26196441
AN - SCOPUS:84952863347
SN - 0022-2836
VL - 427
SP - 3491
EP - 3500
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 22
ER -