The GTP binding protein Obg homolog ObgE is involved in ribosome maturation

Obg proteins belong to a subfamily of GTP binding proteins, which are highly conserved from bacteria to human. Mutations of obgE genes cause pleiotropic defects in various species but the function remained unclear. Here we examine the function of ObgE, the Obg homolog in Escherichia coli. The growth rate correlates with the amount of ObgE in cells. Co‐fractionation experiments further suggest that ObgE binds to 30S and 50S ribosomal subunits, but not to 70S ribosome. Pull‐down assays suggest that ObgE associates with several specific ribosomal proteins of 30S and 50S subunits, as well as RNA helicase CsdA. Purified ObgE cosediments with 16S and 23S ribosomal RNAs in vitro in the presence of GTP. Finally, mutation of ObgE affects pre‐16Sr‐RNA processing, ribosomal protein levels, and ribosomal protein modification, thereby significantly reducing 70S ribosome levels. This evidence implicates that ObgE functions in ribosomal biogenesis, presumably through the binding to rRNAs and/or rRNA‐ribosomal protein complexes, perhaps as an rRNA/ribosomal protein folding chaperone or scaffold protein.

[1]  W. Haldenwang,et al.  Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes. , 2004, Biochemical and biophysical research communications.

[2]  S. M. Sullivan,et al.  The Escherichia coli GTPase CgtAE Cofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase , 2004, Journal of bacteriology.

[3]  M. Dreyfus,et al.  CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit. , 2004, Nucleic acids research.

[4]  D. A. Thayer,et al.  The Caulobacter crescentus CgtAC Protein Cosediments with the Free 50S Ribosomal Subunit , 2004, Journal of bacteriology.

[5]  M. Inouye,et al.  The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli. , 2003, Journal of molecular biology.

[6]  K. Nierhaus,et al.  DnaK-facilitated ribosome assembly in Escherichia coli revisited. , 2003, RNA.

[7]  Arlen W. Johnson,et al.  The Putative GTPases Nog1p and Lsg1p Are Required for 60S Ribosomal Subunit Biogenesis and Are Localized to the Nucleus and Cytoplasm, Respectively , 2003, Molecular and Cellular Biology.

[8]  M. Dreyfus,et al.  The DEAD‐box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli , 2003, Molecular microbiology.

[9]  H. Mori,et al.  [Proteome analysis using MALDI-TOF MS]. , 2003, Seikagaku. The Journal of Japanese Biochemical Society.

[10]  K. Asai,et al.  Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis. , 2002, Microbiology.

[11]  C. Lima,et al.  Structural and biochemical analysis of the Obg GTP binding protein. , 2002, Structure.

[12]  H. Mori,et al.  Genome‐wide analysis of deoxyadenosine methyltransferase‐mediated control of gene expression in Escherichia coli , 2002, Molecular microbiology.

[13]  Wayne A. Decatur,et al.  rRNA modifications and ribosome function. , 2002, Trends in biochemical sciences.

[14]  I. Moll,et al.  Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD‐box helicase on translation of leaderless and canonical mRNAs in Escherichia coli , 2002, Molecular microbiology.

[15]  U. Jakob,et al.  Overexpression of Two Different GTPases Rescues a Null Mutation in a Heat-Induced rRNA Methyltransferase , 2002, Journal of bacteriology.

[16]  G. Węgrzyn,et al.  Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein. , 2002, The Biochemical journal.

[17]  Detlef D. Leipe,et al.  Classification and evolution of P-loop GTPases and related ATPases. , 2002, Journal of molecular biology.

[18]  S. Moriya,et al.  Deficiency of essential GTP‐binding protein ObgE in Escherichia coli inhibits chromosome partition , 2001, Molecular microbiology.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  Akira Ishihama,et al.  Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[21]  W. Haldenwang,et al.  The Bacillus subtilis GTP Binding Protein Obg and Regulators of the ςB Stress Response Transcription Factor Cofractionate with Ribosomes , 2000, Journal of bacteriology.

[22]  H. Niki,et al.  Bidirectional migration of SeqA‐bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[23]  T Horiuchi,et al.  Functional genomics of Escherichia coli in Japan. , 2000, Research in microbiology.

[24]  Patrick Linder,et al.  Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[25]  R. Peery,et al.  16S rRNA Is Bound to Era ofStreptococcus pneumoniae , 1999, Journal of bacteriology.

[26]  W. Haldenwang,et al.  Obg, an Essential GTP Binding Protein ofBacillus subtilis, Is Necessary for Stress Activation of Transcription Factor ςB , 1999, Journal of bacteriology.

[27]  D. Court,et al.  Crystal structure of ERA: a GTPase-dependent cell cycle regulator containing an RNA binding motif. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Wada,et al.  Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE‐bound iterons inhibits DNA replication of mini‐F plasmid in Escherichia coli , 1999, The EMBO journal.

[29]  K. Ochi,et al.  An essential GTP‐binding protein functions as a regulator for differentiation in Streptomyces coelicolor , 1998, Molecular microbiology.

[30]  J. Maddock,et al.  Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins , 1997, Journal of bacteriology.

[31]  M. Sekiguchi.  Genes to cells: edited by Jun-ichi Tomizawa, Blackwell Science Ltd. Institutional: £218.00 (Europe), £242.00 (Rest of World), US$382.00 (USA and Canada). Individual: £65.00 (Europe), £72.00 (Rest of World), US$114.00 (USA and Canada) ISSN 1356 9597 , 1997 .

[32]  M. Pak,et al.  Mechanism of protein remodeling by ClpA chaperone. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A. Vysokanov.  Synthesis of chloramphenicol acetyltransferase in a coupled transcription‐translation in vitro system lacking the chaperones DnaK and DnaJ , 1995, FEBS letters.

[34]  A. Grossman,et al.  Possible role for the essential GTP-binding protein Obg in regulating the initiation of sporulation in Bacillus subtilis , 1995, Journal of bacteriology.

[35]  A. Steinkasserer,et al.  PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations. , 1995, BioTechniques.

[36]  C. Wada,et al.  DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region , 1995, Journal of bacteriology.

[37]  J. Hoch,et al.  Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg , 1994, Journal of bacteriology.

[38]  J. Hoch,et al.  Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis , 1994, Journal of bacteriology.

[39]  J. Alix,et al.  Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Remme,et al.  Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase. , 1993, Journal of molecular biology.

[41]  K. Nierhaus,et al.  The assembly of prokaryotic ribosomes. , 1991, Biochimie.

[42]  K. Rudd,et al.  deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2 , 1991, Journal of bacteriology.

[43]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[44]  K. Isono,et al.  Characterization of the generimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 inEscherichia coli K12 , 1989, Molecular and General Genetics MGG.

[45]  J. Hoch,et al.  The Bacillus subtilis spo0B stage 0 sporulation operon encodes an essential GTP-binding protein , 1989, Journal of bacteriology.

[46]  A. Wada,et al.  Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis. I. Detection of four new proteins. , 1986, Journal of biochemistry.

[47]  H. Aiba.  Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein. , 1985, The Journal of biological chemistry.

[48]  B. Wittmann-Liebold,et al.  The primary structure of protein S5 from the small subunit of the Escherichia coli ribosome , 1978, FEBS letters.

[49]  R. Young,et al.  Complementary sequences 1700 nucleotides apart form a ribonuclease III cleavage site in Escherichia coli ribosomal precursor RNA. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. Brosius,et al.  Occurrence of methylated amino acids as N-termini of proteins from Escherichia coli ribosomes. , 1977, Journal of molecular biology.

[51]  M. Yaguchi.  Primary structure of protein S18 from the small Escherichia coli ribosomal subunit , 1975, FEBS letters.

[52]  R. Cox.  Ribosomes , 1975, Nature.

[53]  L. Lindahl.  Intermediates and time kinetics of the in vivo assembly of Escherichia coli ribosomes. , 1975, Journal of molecular biology.

[54]  W. Held,et al.  Reconstitution of Escherichia coli 30 S ribosomal subunits from purified molecular components. , 1973, The Journal of biological chemistry.

[55]  R. Laursen,et al.  The primary structure of an acidic protein from 50-S ribosomes of Escherichia coli which is involved in GTP hydrolysis dependent on elongation factors G and T. , 1973, European journal of biochemistry.

[56]  G. Mora,et al.  The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. , 1969, Biochemistry.

[57]  P. Traub,et al.  Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[58]  Jay D Keasling,et al.  Effect of lacYExpression on Homogeneity of Induction from the Ptac and Ptrc Promoters by Natural and Synthetic Inducers , 2002, Biotechnology progress.

[59]  H. Noller,et al.  Ribosomes and translation. , 1997, Annual review of biochemistry.

[60]  R. Gourse,et al.  rRNA transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli. , 1996, Annual Review of Microbiology.

[61]  D. Schlessinger,et al.  Mechanism and regulation of bacterial ribosomal RNA processing. , 1990, Annual review of microbiology.

[62]  K. Isono,et al.  Characterization of the gene rimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12. , 1989, Molecular & general genetics : MGG.

[63]  A. Wada.  Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis. II. Characterization of four new proteins. , 1986, Journal of biochemistry.

[64]  J. Steitz,et al.  Identification of a sex-factor-affinity site in E. coli as gamma delta. , 1981, Cold Spring Harbor symposia on quantitative biology.