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F Keppel

Publications and source records attributed to F Keppel.

11 recordsLinked to original sources
PubMed2000

Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31.

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.

Amino Acid Sequence↗
PubMed2000

Genetic analysis of bacteriophage-encoded cochaperonins.

Early genetic studies identified the Escherichia coli groES and groEL genes because mutations in them blocked the growth of bacteriophages lambda and T4. Subsequent genetic and biochemical analyses have shown that GroES and GroEL constitute a chaperonin machine, absolutely essential for E. coli growth, because it is needed for the correct folding of many of its proteins. In spite of very little sequence identity to GroES, the bacteriophage T4-encoded Gp31 protein and the bacteriophage RB49-encoded CocO protein are bona fide GroEL cochaperonins, even capable of substituting for GroES in E. coli growth. A major functional distinction is that only Gp31 and CocO can assist GroEL in the correct folding of Gp23, the major bacteriophage capsid protein. Conserved structural features between CocO and Gp31, which are absent from GroES, highlight their potential importance in specific cochaperonin function.

Bacterial Proteins↗
PubMed1999

Compensatory changes in GroEL/Gp31 affinity as a mechanism for allele-specific genetic interaction.

Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene 31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene 31 that affect interaction with GroEL; all mutations result in changes in the mobile loop of Gp31. Biochemical analyses reveal that the mobile loop mutations alter the affinity between Gp31 and GroEL, most likely by modulating the stability of the GroEL-bound hairpin conformation of the mobile loop. Surprisingly, mutations in groEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL intermediate domain, distantly located from the mobile loop binding site. The observed patterns of genetic and biochemical interaction between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the protein-protein interaction. Mutations studied in this work indirectly alter affinity by modulating a folding transition in the Gp31 mobile loop or by modulating a hinged conformational change in GroEL.

Alleles↗
PubMed1998

The oligomeric structure of GroEL/GroES is required for biologically significant chaperonin function in protein folding.

Two models are being considered for the mechanism of chaperonin-assisted protein folding in E. coli: (i) GroEL/GroES act primarily by enclosing substrate polypeptide in a folding cage in which aggregation is prevented during folding. (ii) GroEL mediates the repetitive unfolding of misfolded polypeptides, returning them onto a productive folding track. Both models are not mutually exclusive, but studies with the polypeptide-binding domain of GroEL have suggested that unfolding is the primary mechanism, enclosure being unnecessary. Here we investigate the capacity of the isolated apical polypeptide-binding domain to functionally replace the complete GroEL/GroES system. We show that the apical domain binds aggregation-sensitive polypeptides but cannot significantly assist their refolding in vitro and fails to replace the groEL gene or to complement defects of groEL mutants in vivo. A single-ring version of GroEL cannot substitute for GroEL. These results strongly support the view that sequestration of aggregation-prone intermediates in a folding cage is an important element of the chaperonin mechanism.

Animals↗
PubMed1990

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein.

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

Amino Acid Sequence↗
PubMed1986

Transcribed human ribosomal RNA genes are attached to the nuclear matrix.

Nuclear matrices were prepared from HeLa nuclei in high or low-salt buffers. After digestion with restriction enzymes, the DNA associated with the nuclear matrix was analysed for its content of ribosomal DNA sequences (rDNA). In both salt conditions, the entire rDNA repeat unit was found to be enriched in the matrix DNA. No enrichment, however, was detected in the absence of rRNA transcription either due to treatment with actinomycin D or when cells arrested in the G0 stage were used to prepare matrices. rDNA was found to be linked to chromosomal scaffolding structures when similar experiments were performed on chromosomes. Moreover, a gradient of enrichment along the rDNA repeat unit was observed, in which the promoter-containing portion was the most highly enriched. I conclude from these experiments that the tandem repeats of rDNA are not randomly associated with matrix or scaffold structure but are probably attached at transcription complexes.

Autoradiography↗
PubMed1980

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function.

Escherichia coli mutants, called groNB, which block the growth of bacteriophage lambda at the level of action of the gene N product, have been isolated as survivors at 42 degrees C of bacteria carrying a) the defective prophage lambda bio11 i lambda cI857 delta H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage lambda cI857. In addition, groNB bacterial mutants have been isolated at 37 degrees C, as large colony formers in the presence of lambda i lambda cI h434, lambda i lambda cI h lambda, and lambda i lambda cI h80 phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42 degrees C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of lambda growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro+ for lambda growth. Lambda transducing phages carrying the groNB+ bacterial gene have been isolated. GroNB mutant bacteria lysogenized by the transducing phage acquire the Gro+ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.

Bacteriophage lambda↗
PubMed1979

Biochemical properties and localization of the chromosomal protein IP25.

The protein IP25, which has previously been reported to accumulate in the chromatin during erythroid differentiation of Friend-virus-transformed erythroleukemia cells (FL cells), is shown to behave like histone H1 without being structurally related to it. Like H1, IP25 is not released by digestion of FL cells nuclei with DNAse I. After micrococcal digestion IP25 and H1 are differentially distributed in the nucleosome monomers and dimers. This distribution suggests an internucleosomal location for IP25 and H1. Different rates of digestion are observed between nuclei of differentiating and non-differentiating FL cells with both DNAse I and micrococcal nuclease. These differences could be due to the presence of IP25 in the chromatin of differentiating cells.

Cell Nucleus↗
PubMed1977

Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells.

Chromatin proteins from erythroleukemia cells transformed by Friend virus were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A new chromosomal protein, apparent molecular weight 25,000, was shown to appear during erythroid differentiation of the cells induced by dimethyl sulfoxide or hexamethylenebisacetamide. This protein is tightly bound to the chromatin and does not appear to be related to any of the known histones. It is also demonstrated that this protein is not induced in a dimethyl sulfoxide-resistant variant of these cells.

Cell Differentiation↗