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P E March

Publications and source records attributed to P E March.

At least 19 recordsLinked to original sources
PubMed2001

Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.

The GTPases comprise a protein superfamily of highly conserved molecular switches adapted to many diverse functions. These proteins are found in all domains of life and often perform essential roles in fundamental cellular processes. Analysis of data from genome sequencing projects demonstrates that bacteria possess a core of 11 universally conserved GTPases (elongation factor G and Tu, initiation factor 2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of GTPases indicate that a second conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes. An emerging concept suggests that the 11 universal GTPases are either necessary for ribosome function or transmitting information from the ribosome to downstream targets for the purpose of generating specific cellular responses. Furthermore, it is suggested that progenitor GTPases were early regulators of RNA function and may have existed in precursors of cellular systems driven by catalytic RNA. If this is the case, then a corollary of this hypothesis is that GTPases that do not bind RNA arose at a later time from an RNA-binding progenitor that lost the capability to bind RNA.

Bacteria↗
PubMed2000

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene.

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.

Blotting, Western↗
PubMed1999

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G.

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.

Amino Acid Sequence↗
PubMed1998

Molecular genetics of bacterial attachment and biofouling.

Microbial adhesion to animate or inert surfaces is potentially mediated by nonspecific physical or specific ligand-receptor interactions. Growth and survival of the microbial community or biofilm then depends on adaptation to a series of changing environmental milieux. Within the realm of cell-cell interaction, recent advances suggest that flagella, fimbriae and other protein receptors are essential for bacterial attachment to surfaces. There has also been profound progress in the elucidation of genes and molecules necessary for bacterial attachments to surfaces and subsequent biofilm formation.

Bacterial Adhesion↗
PubMed1995

Small clusters of divergent amino acids surrounding the effector domain mediate the varied phenotypes of EF-G and LepA expression.

Elongation factors G, Tu, and related proteins (including LepA) form a distinct subgroup within the GTPase superfamily. This observation is based primarily upon amino acid comparisons of the effector region (G2) of the GTP-binding domain. To examine the functional importance of the highly conserved elongation factor G2 domain a series of chimeric proteins were constructed between Escherichia coli EF-G and Micrococcus luteus EF-G, and between E. coli EF-G and LepA (a protein of unknown function). The M. luteus EF-G/E. coli EF-G hybrid, M. luteus EF-G, and E. coli EF-G efficiently complemented EF-G function in an E. coli strain (PEM101) harbouring a temperature-sensitive mutation in fusA (the gene encoding EF-G). A comparison of the amino acid sequences of the M. luteus EF-G and E. coli EF-G indicated that groups of divergent amino acid residues (amino acids 1-9 and 72-80) were not important for function. LepA and LepA/EF-G chimeric proteins were tested for the ability to complement EF-G function in vivo, for cross-linking to 8-azido-[gamma-32P]-GTP in vitro and for fusidic acid-dependent co-sedimentation with 70S ribosomes. With one exception, all chimeras could be readily cross-linked to azido-GTP in an EF-G-like manner, indicating that hybrid protein construction did not generally result in improperly folded GTP-binding domains. However, the inability of such chimeras to complement EF-G function in vivo indicates that the effector domains are not functionally interchangeable. All LepA/EF-G chimeric proteins were severely defective in fusidic acid-dependent complex formation with 70S ribosomes. A comparison of the amino acid sequences of all three proteins suggests that residues 30-33, 43-48, and 63-66 of E. coli EF-G are important for EF-G specific ribosome-associated function.

Amino Acid Sequence↗
PubMed1995

Cross-species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity.

Era is an essential GTP binding protein in Escherichia coli. Two homologs of this protein, Sgp from Streptococcus mutans and Era from Coxiella burnetii, can substitute for the essential function of Era in E. coli. Site-specific and randomly generated Era mutants which may indicate regions of the protein that are of functional importance are described.

Amino Acid Sequence↗
PubMed1994

In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli.

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.

Alleles↗
PubMed1994

GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli.

Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.

Binding, Competitive↗
PubMed1994

Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation.

The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.

Affinity Labels↗
PubMed1992

Membrane-associated GTPases in bacteria.

Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells. This review focuses on membrane-associated GTPases that have been described in prokaryotes. In bacteria, LepA and NodQ are very similar to protein synthesis elongation factors but apparently have membrane-related functions. The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion. Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability. In spite of similarities to well-studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.

Amino Acid Sequence↗
PubMed1991

A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli.

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40 degrees C but completely repressed at 27 degrees C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTP-binding proteins as important regulators of cell growth and division may be ubiquitous in nature.

Blotting, Western↗
PubMed1990

Characterization of the biochemical properties of recombinant ribonuclease III.

An Escherichia coli double strand specific endoribonuclease, RNase III, was cloned, expressed in large amounts, and purified to homogeneity. Enzyme activity was monitored by assaying fractions for the ability to correctly process exogenous RNA containing specific RNase III cleavage sites. DEAE-Sepharose ion exchange chromatography in the presence of a linear KCl gradient (from 0.02 M to 0.75 M) demonstrated that RNase III exists as two distinct forms. One form elutes at a KCl concentration of 0.13 M and the other elutes at 0.33 M. The presence of stoichiometric amounts of the GTP-binding protein Era during purification results in the conversion of the low salt form into the high salt form. Size exclusion chromatography demonstrated that both forms exist as a dimer in solution. In order to investigate the nature of the dimer, protein cross-linking was performed and cross-linked products were detected by silver staining. The protein-protein dimer can be visualized at protein:cross-linker molar ratios as low as 1:15 within 1 minute of exposure to cross-linker in 0.1 M KCl. Upon addition of substrate RNA to the cross-linking reaction a second form of the protein-protein dimer (with a slightly smaller apparent molecular weight) becomes prominent. Induction of the new form is absolutely dependent upon the addition of substrate mRNA to the reaction mixture. We postulate that the RNase III dimer undergoes a dramatic conformational change upon recognition of RNA which we are able to trap by cross-linking.

Chromatography, Ion Exchange↗
PubMed1988

The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth.

The era gene of Escherichia coli encodes a protein (Era) which is similar to the eucaryotic RAS family of proteins. We report here that purified Era possesses both GTP-binding and GTPase activities. Era is also shown to be loosely associated with the inner membrane of E. coli. Overproduction of Era to 5% of the total cellular protein does not apparently alter either cell growth or cAMP levels. Disruption of the era gene by insertional inactivation is shown to be lethal by construction of a conditional lethal era mutant strain.

Bacterial Proteins↗
PubMed1987

The high-molecular-weight proteins of bovine brain plain synaptic vesicles.

High molecular mass polypeptides (Mr greater than 100,000) of plain synaptic vesicles from bovine cerebral cortex were separated using porous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Four major bands, of Mr 262,000, 249,000, 216,000, and 173,000, were resolved. Investigations into the membrane association of the Mr 216,000 and 173,000 proteins by means of solubilization experiments and Sepharose 4B chromatography indicate that the former is a peripheral protein and the latter is more firmly attached, possibly an integral protein. Finally, the Mr 216,000 protein was shown to be highly enriched in synaptic vesicles compared to other brain subfractions. It thus appears to be specifically associated with synaptic vesicles and therefore may have an important role specific to synaptic vesicle function or structure.

Animals↗
PubMed1986

A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins.

The DNA sequence of a gene (era) located immediately downstream of the gene (rnc) encoding ribonuclease III of Escherichia coli was determined and found to encode a protein of 316 amino acid residues. The amino acid sequence of this protein, Era, has significant similarity to the yeast RAS proteins. Overexpression of the Era protein was achieved and GTP cross-linking experiments demonstrated that the protein was indeed capable of binding GTP, as are the yeast and mammalian ras gene products. These data indicate that ras-related sequences occur not only in eukaryotes but also in prokaryotes.

Amino Acid Sequence↗
PubMed1985

The DNA sequence of the gene (rnc) encoding ribonuclease III of Escherichia coli.

The DNA sequence of a 1,076 base pair BglI-BamHI fragment containing the entire rnc gene for ribonuclease III (RNase III) was determined. An open reading frame of 681 base pairs was found in this region which encodes a protein of 227 amino acid residues (calculated molecular weight = 25,218). When this open reading frame was cloned into a high expression vector, pIN-III, a protein of apparent molecular weight of 26,000 was produced upon induction of the cloned gene. This product accounted for up to 5% of the total cellular protein, and comigrated with purified RNase III. RNase III enzyme activity was induced in parallel with the production of the 26,000 molecular weight protein. A putative promoter was found 170 base pairs upstream from the initiation codon. In the long leader region a very stable stem-bulge-stem structure was found which closely resembles typical RNase III cleavage sites. This structure may be cleaved by RNase III to auto-regulate the expression of the rnc gene.

Base Sequence↗
PubMed1985

Characterization of the lep operon of Escherichia coli. Identification of the promoter and the gene upstream of the signal peptidase I gene.

The DNA sequence flanking the gene (lep) encoding signal peptidase I of Escherichia coli has been determined. The upstream flanking sequence contains a gene (lepA) that encodes a polypeptide of 598 amino acid residues and terminates 18 base pairs from the initiation codon of the lep gene. The position of the lep promoter was determined by both gene fusion with the lacZ gene as well as by S1 nuclease mapping of the lep mRNA to be 73 base pairs upstream from the initiation codon of the lepA gene. The lepA gene was cloned into a high expression vector (pIN-III), and its gene product was identified to be a protein of apparent molecular weight of 76,000. This gene product was preferentially localized in the cytoplasmic membrane and periplasmic fractions upon subcellular fractionation. The DNA sequence immediately downstream of the lep gene contains features consistent with a rho factor-independent transcriptional termination site, indicating that the lep operon encodes only two proteins (lepA and lep).

Amino Acid Sequence↗