Expression of Each Cistron in the <i>gal</i> Operon Can Be Regulated by Transcription Termination and Generation of a <i>galK</i>-Specific mRNA, mK2

Wang, Xun,Ji, Sang Chun,Yun, Sang Hoon,Jeon, Heung Jin,Kim, Si Wouk,Lim, Heon M. American Society for Microbiology 2014 Journal of Bacteriology Vol.196 No.14

<P>The <I>gal</I> operon of <I>Escherichia coli</I> has 4 cistrons, <I>galE</I>, <I>galT</I>, <I>galK</I>, and <I>galM</I>. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. <B>378:</B>318–327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the <I>gal</I> operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3′ ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5′ ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at <I>galE-galT</I> and <I>galT-galK</I> junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5′ ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3′ ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5′ portion of pre-mK2 is likely to become mE2. These results also suggested that <I>galK</I> expression could be regulated through mK2 production independent from natural polarity.</P>

Symposium 2 : Transcriptional Regulation ; Superhelicity of DNA and transcription initiation from the gal promoters

( Heon M. Lim ),( Sankar Adhya ) 한국생화학분자생물학회 (구 한국생화학회) 2001 생화학분자생물학회 춘계학술발표논문집 Vol.2001 No.-

Nonsynonymous Variants in <i>PAX4</i> and <i>GLP1R</i> Are Associated With Type 2 Diabetes in an East Asian Population

Kwak, Soo Heon,Chae, Jeesoo,Lee, Seungbok,Choi, Sungkyoung,Koo, Bo Kyung,Yoon, Ji Won,Park, Jin-Ho,Cho, Belong,Moon, Min Kyong,Lim, Soo,Cho, Young Min,Moon, Sanghoon,Kim, Young Jin,Han, Sohee,Hwang, M American Diabetes Association 2018 Diabetes Vol.67 No.9

<P>We investigated ethnicity-specific exonic variants of type 2 diabetes (T2D) and its related clinical phenotypes in an East Asian population. We performed whole-exome sequencing in 917 T2D case and control subjects, and the findings were validated by exome array genotyping in 3,026 participants. In silico replication was conducted for seven nonsynonymous variants in an additional 13,122 participants. Single-variant and gene-based association tests for T2D were analyzed. A total of 728,838 variants were identified by whole-exome sequencing. Among nonsynonymous variants, PAX4 Arg192His increased risk of T2D and GLP1R Arg131Gln decreased risk of T2D in genome-wide significance (odds ratio [OR] 1.48, P = 4.47 x 10(-16) and OR 0.84, P = 3.55 x 10(-8), respectively). Another variant at PAX4 192 codon Arg192Ser was nominally associated with T2D (OR 1.62, P = 5.18 x 10(-4)). In T2D patients, PAX4 Arg192His was associated with earlier age at diagnosis, and GLP1R Arg131Gln was associated with decreased risk of cardiovascular disease. In control subjects without diabetes, the PAX4 Arg192His was associated with higher fasting glucose and GLP1R Arg131Gln was associated with lower fasting glucose and HbA(1c) level. Gene-based analysis revealed that SLC30A8 was most significantly associated with decreased risk of T2D (P = 1.0 x 10(-4)). In summary, we have identified nonsynonymous variants associated with risk of T2D and related phenotypes in Koreans.</P>

Transglutaminase 2 Expression Predicts Progression Free Survival in Non-Small Cell Lung Cancer Patients Treated with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

Jeong, Jae-Heon,Cho, Byoung Chul,Shim, Hyo Sup,Kim, Hye-Ryun,Lim, Sun-Min,Kim, Se Kyu,Chung, Kyung Young,Islam, S.M. Bakhtiar Ul,Song, Jae Jin,Kim, Soo-Youl,Kim, Joo Hang The Korean Academy of Medical Sciences 2013 JOURNAL OF KOREAN MEDICAL SCIENCE Vol.28 No.7

<P>Transglutaminase 2 (TG2), a cross-linking enzyme, is involved in drug resistance and in the constitutive activation of nuclear factor kappa B (NF-κB). We investigated the association of non-small cell lung cancer (NSCLC) treatment efficacy with TG2 and NF-κB expression in 120 patients: 102 with adenocarcinoma and 18 with other histologic types. All patients underwent surgery; 88 received adjuvant chemotherapy, with 28 receiving platinum-based doublet chemotherapy as first-line treatment and 29 receiving epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. Patients' TG2 and NF-κB expression values were calculated semiquantitatively. The median TG2 value was 50 (range, 0-300) and the median NF-κB value was 20 (range, 0-240). Disease-free survival did not differ between the low- and high-TG2 groups. Among patients who received palliative platinum-based doublet chemotherapy, progression free survival (PFS) was longer in the low-TG2 group than in the high-TG2 group (11.0 vs. 7.0 months; <I>P</I>=0.330). Among those who received EGFR-TKI therapy, PFS was also longer in the low-TG2 group than in the high-TG 2 group (11.0 vs. 2.0 months; <I>P</I>=0.013). Similarly, in EGFR wild-type patients treated with EGFR-TKI, PFS was longer in patients with low TG2 expression (9.0 vs. 2.0 months; <I>P</I>=0.013). TG2 expression levels can predict PFS in patients with NSCLC treated with EGFR-TKI.</P>

An Additional Novel Antimicrobial Resistance Gene Cluster inSalmonellaGenomic Island 1 of aSalmonella entericaSerovar Typhimurium DT104 Human Isolate

Kim, Shukho,Kim, Sung-Hun,Chun, Sung-Guen,Park, Mi-Sun,Lim, Heon M.,Lee, Bok Kwon Mary Ann Liebert 2009 Foodborne pathogens and disease Vol.6 No.4

In vivo와 in vitro에서 DicA 단백질의 온도 의존적 DNA 결합

이연호,윤상훈,임헌만,Lee, Yonho,Yun, Sang Hoon,Lim, Heon M. 한국미생물학회 2019 미생물학회지 Vol.55 No.3

In Escherichia coli, DicA protein is involved in cell division control. DicA protein is known to bind DNA better at $25^{\circ}C$ than at $37^{\circ}C$. However, the molecular cause of the temperature dependent binding is not clear. In this study, we investigated how DicA binds DNA and why its DNA binding activity depends on temperature. An unique in vivo DNA binding assay developed in this laboratory showed that unlike the homologous proteins such as RovA or SlyA, DicA uses its N-terminal domain for DNA binding. The in vivo DNA binding assay of DicA also demonstrated that the temperature-dependent DNA binding activity does not come from Cnu or H-NS that is known to bind DNA better at $25^{\circ}C$ than at $37^{\circ}C$. Electrophoretic Mobility Shift Assay (EMSA), when performed with purified DicA protein, did not show temperature-dependent DicA binding activity. However when EMSA was performed with crude protein from WT E. coli cells, temperature-dependent DicA binding activity was observed, suggesting that there is a factor(s) that confers temperature DNA binding activity of DicA in vivo. 대장균 세포분열 조절에 관여하는 DicA 단백질은 $37^{\circ}C$보다 $25^{\circ}C$에서 DNA에 더욱 잘 결합한다. 그러나 DicA 단백질의 온도의존적 DNA 결합에 대한 분자적 원인은 명확하지 않다. 본 연구에서는 DicA 단백질이 어떻게 DNA에 결합하며, 왜 온도 의존적 결합양상을 보이는지 알아보았다. In vivo DNA 결합 분석 결과 RovA나 SlyA와 같은 DicA의 상동성 단백질과는 달리 DicA는 N 말단에 있는 DNA 결합 도메인을 이용하여 20개의 염기쌍으로 이루어진 dicC 조절자 유전자(Oc)에 결합함을 보여주었다. 또한 in vivo 실험에서 DicA는 $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA에 더 잘 결합하는 것으로 알려진 Cnu 또는 H-NS의 영향을 받지 않고 자체적으로 Oc에서의 온도 의존적 DNA 결합을 보인다. 하지만 정제된 DicA 단백질을 이용한 in vitro binding 실험에서는 온도 의존적 DNA 결합이 관찰되지 않았다. Crude 단백질을 이용한 실험에서 DicA 단백질의 온도 의존적 DNA 결합이 관찰되는 것으로 보아 DicA의 온도 의존적 DNA (Oc) 결합은 crude 단백질내에 존재하는 아직 알려지지 않은 in vivo factor에 의해 일어난다.

Epigenomics(Gene Structure and Regulation) : Establishment of mRNA gradient depends on promoter: an investigation of polarity in gene expression

( Heung Jin Jeon ),( Hee Jung Lee ),( Sang Chun Ji ),( Sang Hoon Yun ),( Heon M. Lim ) 한국생화학분자생물학회 (구 한국생화학회) 2008 생화학분자생물학회 춘계학술발표논문집 Vol.2008 No.-

Two-level inhibition of <i>galK</i> expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the <i>galK</i> gene

Wang, Xun,Ji, Sang Chun,Jeon, Heung Jin,Lee, Yonho,Lim, Heon M. National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.24

<P><B>Significance</B></P><P>Most sRNAs of <I>Escherichia coli</I> function at the 5′ end of the target RNA. Binding of sRNA to the 5′ end of the target RNA induces a ribosome-free zone that causes molecular events such as target RNA degradation and Rho-termination. Results from this study show that Spot 42 enhances Rho-termination at the end of the <I>galT</I> gene, demonstrating for the first time that sRNA could function at the 3′ end of the target RNA. The region where Spot 42 binds overlaps with two other functional cis-acting sites: the ribosome-binding site for <I>galK</I> and the cytosine-rich, guanine-poor region known as the Rho-binding site, suggesting a unique molecular mechanism to enhance Rho-termination occurring at a cistron junction in a multicistronic operon.</P><P>The <I>Escherichia coli gal</I> operon has the structure <I>Pgal-galE-galT-galK-galM</I>. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: <I>galE > galT > galK > galM</I>. However, during late-log growth, type 1 polarity is established in which <I>galK</I> is greater than <I>galT</I>, as follows: <I>galE > galK > galT > galM</I>. We found that type 2 polarity occurs as a result of the down-regulation of <I>galK</I>, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the <I>galK-</I>specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of <I>galT</I>. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated <I>galK</I> down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of <I>galT</I>.</P>

dicA promoter DNA에 붙는 H-NS 단백질에 의한 dicA 유전자의 발현 조절

윤상훈,이연호,임헌만,Yun, Sang Hoon,Lee, Yonho,Lim, Heon M. 한국미생물학회 2019 미생물학회지 Vol.55 No.3

H-NS는 대장균에서 DNA 결합 단백질로 수많은 유전자의 발현에 영향을 주는 것으로 잘 알려져 있다. DicA 단백질은 dicF, dicB의 발현을 억제하여 대장균의 분열을 조절한다. dicA의 발현에 Cnu, H-NS의 관여 여부는 CnuK9E 돌연변이가 $37^{\circ}C$에서 dicA의 발현을 억제하여 대장균이 길게 자라는 현상을 일으키며 처음 알려졌다. 하지만 Cnu와 H-NS 두 단백질이 어떻게 dicA의 발현을 조절하는지에 대한 분자적인 기작 연구는 잘 되어있지 않다. 본 연구에서 H-NS가 dicA와 dicC 유전자의 프로모터 부근에 염기서열 특이적으로 결합하며, $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA 더 잘 결합하는 것을 확인하였다. 그리고 EMSA를 통해 Cnu는 H-NS의 DNA 결합의 oligomeric state를 변화시키는 방식으로 작용하는 것을 보여주었다. In vivo transcription assay와 real time PCR을 통해 H-NS가 제거된 대장균에서 dicA 프로모터 활성이 높아지고, 분열 초기 dicA의 발현이 조절 받지 못하고 증가하는 것으로 보아, H-NS는 dicA의 발현에 억제자로서 기능한다. H-NS binds to promoter DNA and works as a general transcription silencer. DicA protein, by binding to the promoter DNA of dicA, activates dicA expression and at the same time inhibits expression of dicF and dicB, thus, exerting cell division control in Escherichia coli. H-NS complexed with a nucleoid protein Cnu was known to be involved in dicA expression. However, the exact nature of H-NS binding to dicA promoter DNA and the consequences of H-NS binding in expression of dicA is not clear. In this study, we explored the DNA binding activity of H-NS on the promoter DNA of dicA and found that H-NS binding occurs exclusively to the dicA promoter DNA. We never observed, however, H-NS binding at the vicinity of the dicA promoter. Temperature dependent oligomerization of H-NS was observed during DNA binding and the Cnu protein enhances the oligomerization process of H-NS binding. In vivo measurement of dicA expression in an hns deleted strain showed that dicA expression increased. These results demonstrated that H-NS binds specifically to dicA promoter DNA and functions as a transcription silencer.

Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes

Wang, Xun,N, Monford Paul Abishek,Jeon, Heung Jin,Lee, Yonho,He, Jin,Adhya, Sankar,Lim, Heon M. National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.10

<P><B>Significance</B></P><P>Transcription termination by RNA polymerase in prokaryotes is well understood in contrast to similar mechanisms in higher organisms. Despite the in vitro occurrence of two types of demonstrable transcription termination events in prokaryotes at the end of transcription units, they are obscured in vivo in two ways: suppression of termination by traversing of the RNA polymerase through the termination sites when coupled to translation, or by further processing of the actual terminated RNA 3′ ends by RNases, as in eukaryotes.</P><P>Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (<I>i</I>) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (<I>ii</I>) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation–transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3′ ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3′ RNA ends; thus the in vivo 3′ ends do not define termination sites. We predict generation of 3′ ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.</P>