117NISR RESEARCH GRANT
2003 Research Grant

Analysis of the Role of E. coli RNA-binding Protein Hfq in theRegulatory Mechanism of Gene Expression
Masaaki WACHI
Department of Bioengineering, Tokyo Institute of Technology
Research aims
The endoribonuclease RNase E, encoded by an essential
gene rne, plays a major role in cellular RNA metabolism,i.e., maturation of functional RNAs such as rRNA andtRNA, degradation of many mRNAs and processing of ftsZmRNA encoding essential cell division protein FtsZ. RNase
E function is somehow regulated by the RNA binding protein Hfq. In this study, we investigate the role of Hfq in intracellular RNA metabolism catalyzed by RNase E.
Methods
E. coli K-12 strain HAT100 [F ara D(lac-pro) thi zce726::Tn10] and its derivatives HAT103 (the same asHAT100 but rne-1), and HAT113 (the same as HAT100 butrne-1 hfq10::cat) were used in this study. Fully grown E.
coli cultures were serially diluted with 0.85% NaCl andspotted onto L agar plates. Plates were incubated at 30°C,
40.5°C and 42°C. Total cellular RNA was isolated and analyzed by the modified agarose gel electrophoresis andNorthern hybridization. FtsZ protein was detected by using
polyclonal anti-FtsZ antiserum (mouse) as first antibody
and alkaline phosphatase-conjugated anti-mouse IgG antibody as second antibody.

RNase E is responsible for maturation of functionalRNAs. We therefore examined effects of the hfq::cat mutation on the maturation of 16S rRNA, 5S rRNA and tRNA.
In the rne control, the processing of these RNA molecules
occurred completely. In the rne-1 strain, precursor molecules of 16S rRNA, 5S rRNA and tRNAPhe were detected at40.5°C and it became accumulated more at 42°C. Introduction of hfq::cat mutation showed almost no effect on thematuration of these RNA molecules. These results showedthat hfq::cat did not recover the maturation of 16S rRNA,
5S rRNA and tRNAPhe affected by rne-1 mutation.
We then analyzed ftsZ transcripts in rne, rne-1, and rne1 hfq::cat strains by Northern hybridization. Unprocessed
precursor transcripts were detected in the rne-1 mutantstrain at both 40.5°C and 42°C. Introduction of hfq::catmutation did not recover the processing of ftsZ mRNA, asin the cases of the maturation of 16S rRNA, 5S rRNA andtRNAPhe. We also examined the synthesis of FtsZ by Western blotting using anti-FtsZ antibody. In the rne-1 mutantstrain, the amount of FtsZ protein decreased remarkably at
40.5°C and 42°C as reported previously. In the rne-1
hfq::cat mutant strain, it was recovered to the almost similar levels of wild-type cells. These results indicate that
hfq::cat mutation recovered translation of FtsZ proteinfrom unprocessed precursor transcripts without recovering
processing of ftsZ mRNA.

Results
The temperature-sensitive rne-1 mutant strain HAT103
showed temperature-sensitive growth at 40.5°C or higher .
Unexpectedly, colony-forming ability of HAT103 was suppressed by introduction of the hfq::cat mutation, almost
completely at 40.5°C and partially even at 42°C (Figure).
This suggests that Hfq protein is involved in essential reaction(s) catalyzed by RNase E.

Figure. Suppression of temperature-sensitivity of rne-1 mutant by
hfq::cat mutation

Conclusion
We found that the temperature-sensitive growth of therne-1 mutant strain was suppressed by introduction of thehfq::cat mutation. The hfq::cat mutation did not affect thematuration of functional RNAs such as 16S rRNA, 5SrRNA and tRNA, and the processing of fstZ mRNA, but recovered the synthesis of FtsZ protein form unprocessed
precursor mRNA. These results suggest that inviability ofRNase E deficiency is due to a decreased level of the cell
division protein FtsZ. The post-transcriptional regulation byRNase and RNA-binding protein demonstrated in this study
could be applied for the protein expression systems in combination with the conventional transcriptional regulations.