Noncoding RNAs Binding to the Nucleoid Protein HU in Escherichia coli

ABSTRACT Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known.

[1]  H. Ochman,et al.  Genome-wide detection of novel regulatory RNAs in E. coli. , 2011, Genome research.

[2]  E. Gilson,et al.  The BIME family of bacterial highly repetitive sequences. , 1991, Research in microbiology.

[3]  Clifford A. Meyer,et al.  Model-based analysis of tiling-arrays for ChIP-chip , 2006, Proceedings of the National Academy of Sciences.

[4]  D. Kamashev,et al.  The binding motif recognized by HU on both nicked and cruciform DNA , 1999, The EMBO journal.

[5]  G. Storz,et al.  Small RNAs in Escherichia coli. , 1999, Trends in microbiology.

[6]  A. Toussaint,et al.  Multiple defects in Escherichia coli mutants lacking HU protein , 1989, Journal of bacteriology.

[7]  S. Tenenbaum,et al.  Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs. , 2003, Molecular cell.

[8]  Michael K. Gilson,et al.  ASAP, a systematic annotation package for community analysis of genomes , 2003, Nucleic Acids Res..

[9]  Jack D. Keene,et al.  Post-transcriptional operons and regulons co-ordinating gene expression , 2005, Chromosome Research.

[10]  Mark Johnson,et al.  NCBI BLAST: a better web interface , 2008, Nucleic Acids Res..

[11]  J. Rouvière-Yaniv,et al.  The histone‐like protein HU binds specifically to DNA recombination and repair intermediates , 2000, The EMBO journal.

[12]  R. Guimarães,et al.  tRNA-rRNA sequence homologies: Evidence for a common evolutionary origin? , 2005, Journal of Molecular Evolution.

[13]  J. Clément,et al.  A novel intercistronic regulatory element of prokaryotic operons , 1982, Nature.

[14]  F. Imamoto,et al.  Participation of hup gene product in replicative transposition of Mu phage in Escherichia coli. , 1989, Gene.

[15]  H. Aiba,et al.  Evidence for two functional gal promoters in intact Escherichia coli cells. , 1981, The Journal of biological chemistry.

[16]  J. Vogel,et al.  Noncoding RNAs encoded by bacterial chromosomes , 2003 .

[17]  S. Bachellier,et al.  Short palindromic repetitive DNA elements in enterobacteria: a survey. , 1999, Research in microbiology.

[18]  A. Worcel,et al.  On the structure of the folded chromosome of Escherichia coli. , 1972, Journal of molecular biology.

[19]  D. Lewis.  Identification of promoters of Escherichia coli and phage in transcription section plasmid pSA850. , 2003, Methods in enzymology.

[20]  E. Gilson,et al.  Palindromic units are part of a new bacterial interspersed mosaic element (BIME). , 1991, Nucleic acids research.

[21]  F. Gros,et al.  Characterization of a novel, low-molecular-weight DNA-binding protein from Escherichia coli. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Tenenbaum,et al.  Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. , 2002, Methods.

[23]  S. Tenenbaum,et al.  Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Storz,et al.  Identification of novel small RNAs using comparative genomics and microarrays. , 2001, Genes & development.

[25]  D. Pettijohn,et al.  Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli. , 1976, Nucleic acids research.

[26]  L. Claret,et al.  Variation in HU composition during growth of Escherichia coli: the heterodimer is required for long term survival. , 1997, Journal of molecular biology.

[27]  N. Kjeldgaard,et al.  Native Escherichia coli HU protein is a heterotypic dimer , 1979, FEBS letters.

[28]  I. Mukerji,et al.  HU binding to a DNA four-way junction probed by Förster resonance energy transfer. , 2011, Biochemistry.

[29]  F. Imamoto,et al.  Preferential binding of E.coli histone-like protein HUα to negatively supercoiled DNA , 1992 .

[30]  D. Pettijohn,et al.  RNA molecules bound to the folded bacterial genome stabilize DNA folds and segregate domains of supercoiling. , 1974, Cold Spring Harbor symposia on quantitative biology.

[31]  M. Beltrame,et al.  Protein HU binds specifically to kinked DNA , 1993, Molecular microbiology.

[32]  F. Imamoto,et al.  Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA. , 1992, Nucleic acids research.

[33]  E. Gilson,et al.  Structural and functional diversity among bacterial interspersed mosaic elements (BIMEs) , 1994, Molecular microbiology.

[34]  S. Tenenbaum,et al.  A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. , 2002, Molecular biology of the cell.

[35]  P. Brown,et al.  Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast , 2004, PLoS biology.

[36]  E. Gilson,et al.  A family of dispersed repetitive extragenic palindromic DNA sequences in E. coli. , 1984, The EMBO journal.

[37]  R. Hengge-aronis,et al.  The Escherichia coli histone‐like protein HU regulates rpoS translation , 2001, Molecular microbiology.

[38]  D. Pettijohn,et al.  Synthesis of ribosomal RNA on a protein-DNA complex isolated from bacteria: a comparison of ribosomal RNA synthesis in vitro and in vivo. , 1970, Journal of molecular biology.

[39]  H. Niki,et al.  Maintenance of plasmids in HU and 1HF mutants of Escherichia coli , 2004, Molecular and General Genetics MGG.

[40]  D. Court,et al.  Mini-λ: a tractable system for chromosome and BAC engineering , 2003 .

[41]  F. Blattner,et al.  Functional Genomics: Expression Analysis ofEscherichia coli Growing on Minimal and Rich Media , 1999, Journal of bacteriology.

[42]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[43]  C. Guthrie,et al.  Identification of Lhp1p-associated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  G. Storz,et al.  6S RNA Regulates E. coli RNA Polymerase Activity , 2000, Cell.

[45]  A. Mazur,et al.  HU binds and folds single-stranded DNA , 2007, Nucleic acids research.

[46]  G. Storz,et al.  Global analysis of small RNA and mRNA targets of Hfq , 2003, Molecular microbiology.

[47]  H. Margalit,et al.  A survey of small RNA-encoding genes in Escherichia coli. , 2003, Nucleic acids research.

[48]  J. Oberto,et al.  Histones, HMG, HU, IHF: Même combat. , 1994, Biochimie.

[49]  J. Oberto,et al.  Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium , 1996, Journal of bacteriology.

[50]  C. Nicchitta,et al.  Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation. , 2005, Molecular biology of the cell.

[51]  D. Court,et al.  Mini-lambda: a tractable system for chromosome and BAC engineering. , 2003, Gene.

[52]  Aixia Zhang,et al.  An antibody-based microarray assay for small RNA detection , 2006 .

[53]  P. Brown,et al.  Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[54]  R. Haselkorn,et al.  Cyanobacterial DNA-binding protein related to Escherichia coli HU. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[55]  A. Barkan,et al.  RNA Immunoprecipitation and Microarray Analysis Show a Chloroplast Pentatricopeptide Repeat Protein to Be Associated with the 5′ Region of mRNAs Whose Translation It Activatesw⃞ , 2005, The Plant Cell Online.

[56]  C. Guthrie,et al.  Functional specificity of shuttling hnRNPs revealed by genome-wide analysis of their RNA binding profiles. , 2005, RNA.

[57]  D. Kamashev,et al.  The Bacterial Histone-like Protein HU Specifically Recognizes Similar Structures in All Nucleic Acids , 2002, The Journal of Biological Chemistry.

[58]  E. Bonnefoy,et al.  HU and IHF, two homologous histone‐like proteins of Escherichia coli, form different protein‐DNA complexes with short DNA fragments. , 1991, The EMBO journal.

[59]  D. Pettijohn,et al.  The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[60]  K. Swinger,et al.  Structure-based analysis of HU-DNA binding. , 2007, Journal of molecular biology.

[61]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.