RNA-binding specificity of E. coli NusA

The RNA sequences boxA, boxB and boxC constitute the nut regions of phage λ. They nucleate the formation of a termination-resistant RNA polymerase complex on the λ chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the λ N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K_d values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why λ requires an additional protein, λ N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.     

Transcriptional regulation by antitermination. Interaction of RNA with NusB protein and NusB/NusE protein complex of Escherichia coli

A recombinant heterodimeric NusB/NusE protein complex of Escherichia coli was expressed under the control of a synthetic mini operon. Surface plasmon resonance measurements showed that the heterodimer complex has substantially higher affinity for the boxA RNA sequence motif of the ribosomal RNA (rrn) operons of E.coli as compared to monomeric NusB protein. Single base exchanges in boxA RNA reduced the affinity of the protein complex up to 15-fold. The impact of base exchanges in the boxA RNA on the interaction with NusB protein was studied by (1)H,(15)N heterocorrelation NMR spectroscopy. Spectra obtained with modified RNA sequences were analysed by a novel generic algorithm. Replacement of bases in the terminal segments of the boxA RNA motif caused minor chemical shift changes as compared to base exchanges in the central part of the dodecameric boxA motif.     

Lytic Replication of Coliphage Lambda in Salmonella typhosa Hybrids

Hybrids between Escherichia coli K-12 and Salmonella typhosa which conserved a continuous K-12 chromosomal diploid segment extending from pro through ara to the strA locus were sensitive to plaque formation by wild-type λ. These partially diploid S. typhosa hybrids could be lysogenized with λ and subsequently induced to produce infectious phage particles. When the K-12 genes were segregated from a lysogenic S. typhosa hybrid, phage-productive ability was no longer detectable due to loss of a genetic region necessary for vegetative replication of λ. However, λ prophage was shown to persist in a quiescent state in the S. typhosa hybrid segregant with phage-productive ability being reactivated after replacement of the essential K-12 λ replication region. Low-frequency transduction and high-frequency transduction lysates containing the gal⁺ genes of S. typhosa were prepared by induction of λ-lysogenic S. typhosa hybrids indicating that the attλ site is chromosomally located in S. typhosa in close proximity to the gal locus as in E. coli K-12. After propagation in S. typhosa hybrids, λ was subject to restriction by E. coli K-12 recipients, thus establishing that S. typhosa does not perform the K-12 modification of λ deoxyribonucleic acid. Hybrids of S. typhosa, however, did not restrict λ grown previously on E. coli K-12. The K-12 genetic region required for λ phage production in S. typhosa was located within min 66 to min 72 on the genetic map of the E. coli chromosome. Transfer of an F-merogenote encompassing the 66 to 72 min E. coli chromosomal region to λ-insensitive S. typhosa hybrids enabled them to replicate wild-type λ. The λ-insensitive S. typhosa hybrid, WR4255, which blocks λ replication, can be mutagenized to yield mutant strains sensitive to λvir and λimm434. These WR4255 mutants remained insensitive to plaque formation by wild-type λ.     

Bacterial Cell Division Regulation: Lysogenization of Conditional Cell Division lon− Mutants of Escherichia coli by Bacteriophage Lambda

The lon⁻ mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon⁻ mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon⁻ mutants efficiently and consequently forms clear plaques on a lon⁻ host. Two lines of evidence suggest that this failure probably results from interference with expression of the λcI gene, which codes for repressor, or with repressor action:-(i) when a lon⁻ mutant was infected with a λcII, cIII, or c Y mutant, there was an additive effect between the lon⁻ mutation and the λc mutations upon reduction of lysogenization frequency; and (ii) lon⁻ mutants permitted the growth of the λcro⁻ mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon⁻ cells is also reported.     

Temperature-Sensitive Modification and Restriction Phenotypes of an Escherichia coli dnaD Mutant

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of λ phage at 41 C. However, the λ phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the λ phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified λ·C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.     

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030–1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1–P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1–P2)₂, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1–P2 heterodimer is required for subsequent formation of the P0(P1–P2)₂ complex and its functional rRNA binding in silkworm ribosomes.     

Purine Substrate Recognition by the Nucleobase-Ascorbate Transporter Signature Motif in the YgfO Xanthine Permease: ASN-325 BINDS AND ALA-323 SENSES SUBSTRATE*

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315–339 for sensitivity to inactivation by 2-sulfonatoethyl methanethiosulfonate (MTSES⁻) and N-ethylmaleimide (NEM) showed a similar pattern: highly sensitive mutants clustering at the motif sequence (323–329) and a short α-helical face downstream (332, 333, 336). In the presence of substrate, N325C is protected from alkylation with either MTSES⁻ or NEM, whereas sensitivity of A323C to inactivation by NEM is enhanced, shifting IC₅₀ from 34 to 14 μm. Alkylation or sensitivity of the other mutants is unaffected by substrate; the lack of an effect on Q324C is attributed to gross inability of this mutant for high affinity binding. Site-directed mutants G333R and S336N at the α-helical face downstream the motif display specific changes in ligand recognition relative to wild type; G333R allows binding of 7-methyl and 8-methylxanthine, whereas S336N disrupts affinity for 6-thioxanthine. Finally, all assayable motif-mutants are highly accessible to MTSES⁻ from the periplasmic side. The data suggest that the NAT motif region lines the solvent- and substrate-accessible inner cavity, Asn-325 is at the binding site, Ala-323 responds to binding with a specific conformational shift, and Gly-333 and Ser-336 form part of the purine permeation pathway.     

Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro⁺, in λcro⁻ plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro⁻ plasmid replication control.