Kook Han

Roles of rpoS‐activating small RNAs in pathways leading to acid resistance of Escherichia coli

Escherichia coli and related enteric bacteria can survive under extreme acid stress condition at least for several hours. RpoS is a key factor for acid stress management in many enterobacteria. Although three rpoS‐activating sRNAs, DsrA, RprA, and ArcZ, have been identified in E. coli, it remains unclear how these small RNA molecules participate in pathways leading to acid resistance (AR). Here, we showed that overexpression of ArcZ, DsrA, or RprA enhances AR in a RpoS‐dependent manner. Mutant strains with deletion of any of three sRNA genes showed lowered AR, and deleting all three sRNA genes led to more severe defects in protecting against acid stress. Overexpression of any of the three sRNAs fully rescued the acid tolerance defects of the mutant strain lacking all three genes, suggesting that all three sRNAs perform the same function in activating RpoS required for AR. Notably, acid stress led to the induction of DsrA and RprA but not ArcZ.

Rho-dependent Termination of ssrS (6S RNA) Transcription in Escherichia coli IMPLICATION FOR 3′ PROCESSING OF 6S RNA AND EXPRESSION OF DOWNSTREAM ygfA (PUTATIVE 5-FORMYL-TETRAHYDROFOLATE CYCLO-LIGASE)

It is well known that 6S RNA, a global regulatory noncoding RNA that modulates gene expression in response to the cellular stresses in Escherichia coli, is generated by processing from primary ssrS (6S RNA) transcripts derived from two different promoters. The 5′ processing of 6S RNA from primary transcripts has been well studied; however, it remains unclear how the 3′-end of this RNA is generated although previous studies have suggested that exoribonucleolytic trimming is necessary for 3′ processing. Here, we describe several Rho-dependent termination sites located ∼90 bases downstream of the mature 3′-end of 6S RNA. Our data suggest that the 3′-end of 6S RNA is generated via exoribonucleolytic trimming, rather than endoribonucleolytic cleavage, following the transcription termination events. The termination sites identified in this study are within the open reading frame of the downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase) gene, a part of the highly conserved bacterial operon ssrS-ygfA, which is up-regulated during the biofilm formation. Our findings reveal that ygfA expression, which also aids the formation of multidrug-tolerant persister cells, could be regulated by Rho-dependent termination activity in the cell.

Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain.

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNAAsn precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity—implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.

Electrophoretic mobility shift assay of RNA-RNA complexes.

A simple, rapid, and sensitive electrophoretic mobility shift assay (EMSA) can be successfully used to analyze RNA-RNA interactions. The EMSA of RNA-RNA complexes can be further used to evaluate the specificity of interactions using competitor RNAs in combination with their mutated versions or nonspecific RNAs, such as yeast tRNA. RNA is simply prepared by in vitro transcription from PCR product templates. Detailed experimental descriptions for EMSA-based analysis of specific RNA-RNA interactions between Sib RNAs and ibs mRNAs as a representative example are presented.

Structural analysis of Escherichia coli C5 protein

Structural analysis of Escherichia coli C5 protein Jae-Sun Shin, Kwang-Sun Kim, Kyoung-Seok Ryu, Kook Han, Younghoon Lee, and Byong-Seok Choi* 1Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea 2 Biomedical Proteomics Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea 3 Systems and Synthetic Biology Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea 4 Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea 5Division of Magnetic Resonance Research, Korea Basic Science Institute, Yangcheong-Ri 804-1, Ochang-Eup, Cheongwon-Gun, Chungbuk 363-883, Korea