Journal of Microbiology

Journal Article

Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein: Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn; J. Microbiol. 2024;62(10):871-882. Published online September 6, 2024; DOI: https://doi.org/10.1007/s12275-024-00169-2

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The Escherichia coli cAMP receptor protein (CRP) relies on the F-helix, the recognition helix of the helix-turn-helix motif, for DNA binding. The importance of the CRP F-helix in DNA binding is well-established, yet there is little information on the roles of its non-base-contacting residues. Here, we show that a CRP F-helix position occupied by a non-base-contacting residue Val183 bears an unexpected importance in DNA binding. Codon randomization and successive in vivo screening selected six amino acids (alanine, cysteine, glycine, serine, threonine, and valine) at CRP position 183 to be compatible with DNA binding. These amino acids are quite different in their amino acid properties (polar, non-polar, hydrophobicity), but one commonality is that they are all relatively small. Larger amino acid substitutions such as histidine, methionine, and tyrosine were made site-directedly and showed to have no detectable DNA binding, further supporting the requirement of small amino acids at CRP position 183. Bioinformatics analysis revealed that small amino acids (92.15% valine and 7.75% alanine) exclusively occupy the position analogous to CRP Val183 in 1,007 core CRP homologs, consistent with our mutant data. However, in extended CRP homologs comprising 3700 proteins, larger amino acids could also occupy the position analogous to CRP Val183 albeit with low occurrence. Another bioinformatics analysis suggested that large amino acids could be tolerated by compensatory small-sized amino acids at their neighboring positions. A full understanding of the unexpected requirement of small amino acids at CRP position 183 for DNA binding entails the verification of the hypothesized compensatory change(s) in CRP.

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SPD_0410 negatively regulates capsule polysaccharide synthesis and virulence in Streptococcus pneumoniae D39
Ye Tao, Li Lei, Shuhui Wang, Xuemei Zhang, Yibing Yin, Yuqiang Zheng
Frontiers in Microbiology.2025;[Epub] CrossRef

Review

cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor: Hwan Youn , Marcus Carranza; J. Microbiol. 2023;61(3):277-287. Published online March 9, 2023; DOI: https://doi.org/10.1007/s12275-023-00028-6

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Abstract

The active and inactive structures of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcr!ption factor, are compared to generate a paradigm in the cAMP-induced activation of CRP. The resulting paradigm is shown to be consistent with numerous biochemical studies of CRP and CRP*, a group of CRP mutants displaying cAMP-free activity. The cAMP affinity of CRP is dictated by two factors: (i) the effectiveness of the cAMP pocket and (ii) the protein equilibrium of apo-CRP. How these two factors interplay in determining the cAMP affinity and cAMP specificity of CRP and CRP* mutants are discussed. Both the current understanding and knowledge gaps of CRP-DNA interactions are also described. This review ends with a list of several important CRP issues that need to be addressed in the future.

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Identification of a cellular role of hemolysin co-regulatory protein (Hcp) in Vibrio alginolyticus modulating substrate metabolism and biofilm formation by cAMP-CRP
Shuilong Wu, Yu Huang, Minhui Wu, Huapu Chen, Bei Wang, Kwaku Amoah, Jia Cai, Jichang Jian
International Journal of Biological Macromolecules.2024; 282: 136656. CrossRef
cAMP-independent DNA binding of the CRP family protein DdrI from Deinococcus radiodurans
Yudong Wang, Jing Hu, Xufan Gao, Yuchen Cao, Shumai Ye, Cheng Chen, Liangyan Wang, Hong Xu, Miao Guo, Dong Zhang, Ruhong Zhou, Yuejin Hua, Ye Zhao, Paul Babitzke
mBio.2024;[Epub] CrossRef
Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein
Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn
Journal of Microbiology.2024; 62(10): 871. CrossRef
Bacterial Regulatory Mechanisms for the Control of Cellular Processes: Simple Organisms’ Complex Regulation
Jin-Won Lee
Journal of Microbiology.2023; 61(3): 273. CrossRef
Mechanisms and biotechnological applications of transcription factors
Hehe He, Mingfei Yang, Siyu Li, Gaoyang Zhang, Zhongyang Ding, Liang Zhang, Guiyang Shi, Youran Li
Synthetic and Systems Biotechnology.2023; 8(4): 565. CrossRef

Journal Articles

Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity: Young-Il Kim , Mark Anthony B. Casel , Se-Mi Kim , Seong-Gyu Kim , Su-Jin Park , Eun-Ha Kim , Hye Won Jeong , Haryoung Poo , Young Ki Choi; J. Microbiol. 2020;58(10):886-891. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0335-6

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Abstract

Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The
results
substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful
method
for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

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Sankar Prasad Chaki, Melissa M. Kahl-McDonagh, Benjamin W. Neuman, Kurt A. Zuelke
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Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction: Matt N. Hicks , Sanjiva Gunasekara , Jose Serate , Jin Park , Pegah Mosharaf , Yue Zhou , Jin-Won Lee , Hwan Youn; J. Microbiol. 2017;55(10):816-822. Published online September 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7266-x

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Abstract

The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP’s recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

Citations

Citations to this article as recorded by

cAMP-independent DNA binding of the CRP family protein DdrI from Deinococcus radiodurans
Yudong Wang, Jing Hu, Xufan Gao, Yuchen Cao, Shumai Ye, Cheng Chen, Liangyan Wang, Hong Xu, Miao Guo, Dong Zhang, Ruhong Zhou, Yuejin Hua, Ye Zhao, Paul Babitzke
mBio.2024;[Epub] CrossRef
Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein
Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn
Journal of Microbiology.2024; 62(10): 871. CrossRef
cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor
Hwan Youn, Marcus Carranza
Journal of Microbiology.2023; 61(3): 277. CrossRef

Research Support, Non-U.S. Gov't

Saccharomyces cerevisiae Cmr1 Protein Preferentially Binds to UV-Damaged DNA In Vitro: Do-Hee Choi , Sung-Hun Kwon , Joon-Ho Kim , Sung-Ho Bae; J. Microbiol. 2012;50(1):112-118. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1597-4

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Abstract: DNA metabolic processes such as DNA replication, recombination, and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study, we isolated DNA-binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing the proteins using mass spectrometry, an uncharacterized protein, Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C-terminal WD40 repeats. Consistent with this finding, the purified recombinant Cmr1 protein was found to be intrinsically associated with DNA-binding activity and exhibited higher affinity to UV-damaged DNA substrates. Chromatin isolation experiments revealed that Cmr1 localized in both the chromatin and supernatant fractions, and the level of Cmr1 in the chromatin fraction increased when yeast cells were irradiated with UV. These
results
suggest that Cmr1 may be involved in DNA-damage responses in yeast.

An Analysis of the Arm-type Site Binding Domain of Bacteriophage γ Integrase: Cho , Eun Hee; J. Microbiol. 1995;33(2):165-170.

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Abstract: The 356 amino acid long lambda integrase protein of bacteriophage λ constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P_tac promoter and the lacI^q gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

Binding of IciA Protein to the dnaA Promoter Region: Kim, Hak Jung , Hwang, Deog Su; J. Microbiol. 1995;33(3):191-195.

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Abstract: IciA protein has been shown as an inhibitor for the initiation of E. coli chromosomal DNA replication at oriC. IciA protein binds the AT-rich region in oriC and then blocks the initiation of chromosomal DNA replication. Two binding sites for IciA protein were identified in dnaA gene, encoding the initiator for the E. coli chromosomal replication, promoter region by gel-shift assay and DNase I footprinting, One, named as IciA site I, is located upstream of the dnaA promoter 1P. The other, named as IciA site II, is located downstream of the dnaA promoter 2P. The sequence comparison of the regions protected from the DNase I cleavage did not result in a clear consensus sequence for the binding of IciA protein, suggesting that IciA protein may be a member of multimeric complex dsDNA binding proteins. This study provided information about the binding mode of IciA protein. Even though the IciA site II and IciA binding site in oriC seem to be composed of two IciA binding units, one binding unit is likely enough to cause the binding of IciA protein to the IciA site I. The binding of IciA protein to the dna4 promoter implies that IciA protein may involve not only the control of the initiation of chromosomal DNA replication but also the control of the dna4 gene expression.

Secondary Structure Analysis of Amino Terminal Domain in Phage Lambda Integrase: Yu, Jeong A , Nam, Chan Eun , Cho, Eun Hee; J. Microbiol. 1998;36(4):266-272.

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Abstract: The amino-terminal domain of bacteriophage λ integrase recognizes specific DNA sequences called arm-type sites. To study the structural and functional relationships of the integras armtype DNA binding domains were confirmed by gel mobility-shift assay. The polypeptides were subjected to circular dichroism spectroscopy to estimate the amount of secondary structures they contain Based upon analyses of circular dichroism spectra and comparison with predicted secondary structural compositions, it was estimated that the amino terminal domain of integrase in an aqueous solution was composed of a little α-helical region. The helical content increased with an increasing amount of ethanol, an α-helix inducer. This indicates that its conformation can be changed to a form with higher content of α-helical structure under a certain condition.

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