Werner K. Maas

Amyloid-like interactions within nucleoporin FG hydrogels.

The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the N-terminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular β-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain β-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.

STUDIES ON THE MECHANISM OF REPRESSION OF ARGININE BIOSYNTHESIS IN ESCHERICHIA COLI. II. DOMINANCE OF REPRESSIBILITY IN DIPLOIDS.

Strains have been isolated which are diploid for about one-third of the linkage map of E. coli, including a locus Rarg which controls repressibility of the enzymes of arginine biosynthesis. In Rarg+/Rarg− merodiploids, isolated from a cross of a repressible (Rarg+) double-male donor with a non-repressible (Rarg−) recipient, arginine is able to repress enzyme formation, showing that repressibility, as in Rarg+/Rarg− merozygotes, is a dominant trait. In interpreting this finding it is suggested that the gene Rarg+ controls the production of a specific repressor in the cytoplasm. This interpretation has been given previously for similar findings in other metabolic pathways, notably for the control of the enzymes of lactose fermentation and of histidine and tryptophan biosynthesis. The term regulon is proposed to describe such a system in which the production of all enzymes can be controlled by a single repressor substance. Two types of regulons can be distinguished, those in which the structural genes for the enzymes are adjacent to each other (histidine, tryptophan) and which thus consist of single operons, and those in which they are not (arginine) and which thus consist of several operons.

Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E. coli B

We have found a branched DNA-RNA compound in E. coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria. This compound is not produced in E. coli K12. We have cloned a 3.5 kb chromosomal segment of E. coli B, which, when transferred into E. coli K12, leads to the production of the DNA-RNA compound. We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation. The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase. This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro.

Binding of the arginine repressor of Escherichia coli K12 to its operator sites

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

Mutational analysis of the arginine repressor of Escherichia coli

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild‐type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N‐terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by l‐arginine at the same concentration as the wild‐type repressor. The other two mutant repressors map in the C‐terminal half of the protein. They are poorly precipitated by L‐arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super‐repressors. Such mutants behave as arginine auxotrophs as a result of hyper‐repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super‐repressor proteins were purified, in comparison with the wild‐type repressor, two of them were shown to have a higher DNA‐binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA‐binding affinity when bound to arginine.

Multicopy single‐stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein

Multicopy single‐stranded DNA (msDNA) molecules consist of single‐stranded DNA covalently linked to RNA. In Escherichia coli, such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem‐loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli. To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Studies on the mechanism of repression of arginine biosynthesis in Escherichia coli: III. Repression of enzymes of arginine biosynthesis in arginyl-tRNA synthetase mutants†*

Canavanine-resistant mutants of Escherichia coli K12 have been isolated which produce a defective arginyl-tRNA synthetase, the activity of the enzyme in various mutants ranging from about 3 to 50% of that in the wild type. On the basis of their growth rates in the presence and absence of arginine, the mutants can be put into two groups. Class I mutants (slow growth in the absence of arginine, normal growth in the presence of arginine) have an altered arginyl-tRNA synthetase with a decreased affinity for arginine. No marked changes were noticed in the enzyme obtained from class II mutants (slow growth in the absence or presence of arginine). Repression by arginine of the enzymes of arginine synthesis, as measured by the rate of formation of ornithine transcarbamylase and argininosuccinase, was not impaired in either class. Levels of charged tRNAArg in growing cells showed no correlation between the state of repression and the degree of charging. Thus for each strain tested there was no appreciable difference in the level of charging between repressed cells, growing with arginine, and de-repressed cells, growing without arginine. For class II mutants this level was about 20% of the total arginine acceptor capacity of tRNA, for class I mutants about 75%, and for the parent strains with a normal arginyl-tRNA synthetase, 80 to 90%. We conclude that the bulk of tRNAArg is not involved in repression, but we have not ruled out participation of a small fraction of this tRNA.

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.

Multicopy single‐stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli

Retrons are genetic elements that encode multicopy single‐stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single‐stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem‐loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem‐loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.