Mutsuo Imai

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

SummaryWithin the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Mutant rho factors with increased transcription termination activities. II: Identification and functional dissection of amino acid changes

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.

Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

SummaryTwo-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam− strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam− strand with the Dam+ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.

Studies on the altered rho factor in nitA mutants of Escherichia coli defective in transcription termination: I. Characterization and quantitative determination of rho in cell extracts

Abstract Rho factors in cell extracts from transcription termination-defective strains nitA18, nitA702 (temperature-sensitive) and nitA112 (amber type) have been quantitatively characterized by immunological reactions and electrophoretic analysis on sodium dodecyl sulphate/polyacrylamide gels. Both the antigenicity and molecular size (the subunit peptide) of these mutant rho (nitA112 rho under suppressed conditions) are apparently identical with those of the wild-type. However, the rho factors in the nitA18 and nitA702 extracts are quite sensitive to proteolytic inactivation. Cellular contents of these two mutant rho also differ from those of the wild-type rho (0.1 to 0.15% of the total protein). The content of the nitA702 rho is more than doubled at a permissive temperature, and is further increased severalfold at a non-permissive temperature. This implies that an autogenous regulation is operating in the cellular synthesis of rho. In the nitA112 mutant, which carries the temperature-sensitive amber suppressor, production of the altered rho is much less than normal at a permissive temperature, and is barely detectable at a non-permissive temperature where an amber fragment of the rho peptide appears instead of the intact rho. These results provide evidence that the rho factor is a direct product of the nitA gene.

The use of operon fusions in studies of the heat-shock response: effects of altered sigma 32 on heat-shock promoter function in Escherichia coli.

SummaryDerivatives of λpF13 phage in which lacZ expression (β-galactosidase synthesis) is directed by transcription initiated at a heat-shock promoter (PrpoDhs or PgroE) were constructed and used for analysis of the heat-shock response in Escherichia coli. A wild-type strain (MC4100) lysogenic for either of these phages exhibited typical transient induction of β-galactosidase synthesis upon a temperature shift from 30° to 42° C or after addition of ethanol to the medium (4% to 5%) at 30° C. In contrast, most amber rpoH (htpR) mutants tested (in a Su- background) failed to respond to a temperature shift, though some mutants affected in the carboxy-terminal region exhibited a partial response. All rpoH mutants tested showed a weak but significant response to ethanol. F′ plasmids carrying each of six known nonsense suppressors were then introduced into each of four rpoH amber mutants lysogenic for λpF13-(Phs-lacZ), creating a set of F′ strains that produce sigma 32 protein with a specific amino acid substitution at a known site. Some of these strains showed an essentially normal heat-shock response while others showed little response with either or both of the promoters. In some instances, the response was significantly delayed. These results point to the usefulness of the λpF13-derivative phages for quantitative and systematic analysis of heat-shock response in E. coli.

Studies on the altered rho factor in nitA mutants of Escherichia coli defective in transcription termination. II. Purification and molecular properties of the mutant rho.

Abstract The transcription-termination factor rho was purified from two termination-defective strains, nitA18 and nitA702 (temperature-sensitive), by the method of Roberts (1969) as modified due to the alteration of their molecular properties. The mutant rho factors are quite sensitive to temperature and proteolysis even after purification. The nitA702 rho shows a reduced affinity for phosphocellulose and a lower sedimentation rate (5.5 S) than does the wild-type (9.5 S). The transcription-termination activity is significantly decreased in both mutant rho factors; the maximum inhibition by the nitA18 and nitA702 rho being limited to 60% and 15%, respectively, whereas the wild-type inhibits 65% or more at 37 °C. The poly(C)-dependent ATPase activities (Richardson et al., 1975) of these mutant rho factors are apparently normal at 37 °C or below. However, they showed altered enzyme specificity with activator RNAs containing ribonucleosides other than cytidine. The ATPase activity of the nitA702 rho is much less than normal when activated with the nascent RNA produced by the transcription in vitro of various DNAs or with such synthetic ribohomopolymers as poly(A), poly(U) and poly(I). In contrast, the nitA18 rho exhibits significantly higher ATPase activity with all these RNA activators, in spite of its decreased activity in transcription termination. The altered functions of these mutant rho in transscription-termination regulation are discussed.

Isolation and genetic characterization of the nitA mutants of Escherichia coli affecting the termination factor rho.

SummaryTaking advantage of the Spi (Sensitivity to P2 interference) phenomenon, bacterial mutants seemingly resistant to phage λsusN7nin5, but sensitive to phage λspi, were isolated from a strain of E. coli K12 carrying no nonsense suppressor and lysogenic for P2. A class of these mutants, designated nitA (N-independent transcription), is described here.Upon infection of the nitA mutants with a trp transducing phage λsusN7N53ptrp46 which carries the E. coli trpE and D genes in the CIII-att region of the λ genome, formation of anthranilate synthetase (ASase, a complex protein of trp E and D gene products) was clearly demonstrated. In contrast, no ASase formation was observed in the parent nitA+strain under the same conditions. The synthesis is subject to “turn off” control, and is completely repressed by the CI repressor of phage λ. The nitA cells lysogenic for λCI857susN7N53 are killed by thermal induction much more efficiently than the parent cells lysogenic for the same phage. The nitA mutants support the growth of λsusN7N53byp much better than the parent. These results suggest that the nitA mutation permits the early leftward and rightward transcription of the λ genome in the absence of the N gene product.On the E. coli genetic map, nitA is located between ilv and metE, nearer to ilv. The mutant allele is recessive to the wild-type allele. The present evidence, together with results of biochemical investigations to be reported, suggests that nitA is a gene specifying the transcription termination factor rho.

Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda

SummaryTermination of transcription at bacteriophage λ terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E. coli. Experiments using terminator-assay λ vectors revealed that the efficiency of termination at both ρ-dependent (λtL1) and ρ-independent (λtL2 and trp a) terminators decreases in the mutant. The mutation does not block λ phage growth at either permissive or nonpermissive temperatures, nor does it affect the λ Q protein antitermination activity at the t6S terminator. These results indicate that NusA is required for transcription termination, and that λ N and Q-mediated antitermination may not require the NusA protein function in the nusAts11 mutant.

Isolation and characterization of rho mutants of Escherichia coli with increased transcription termination activities.

SummaryA novel type of rho mutants, rhos, with increased transcription termination activities have been isolated. A termination defective rho mutation rho-ts702 (formerly designated nitA702), which causes temperature-sensitive cell growth, was found to be dominant over the wild-type allele in relieving mutational polarity. The rhos mutations were derived as temperature-resistant revertants of rho-ts702 carried by λ transducing phage. They exhibited dominance over rho-ts702 leading to restoration of polarity. When the rhos mutations were introduced into the Escherichia coli chromosome, they caused increased polarity in the trp and lac operons. The rhos mutants were classified into two groups in terms of their terminator specificity: The first group demonstrated increased termination efficiencies against all terminators tested, whereas the second exhibited various efficiencies, either more than or less than the normal level depending on the terminator. The cellular content of ϱ protein in each rhos strain was significantly lower than that in the rho+ strain. Moreover, in an in vitro transcription system, purified ρs proteins showed increased termination activities against the trpE pseudoterminators. These results indicate that the rhos phenotype is due to qualitative alterations, rather than quantitative increases, of the ϱ protein. The reduced content of ρs enforces the current notion that the rho gene is autogenously regulated by rho-dependent transcriptional attenuation.

Regulation of the tryptophan operon of Escherichia coli integrated into the phage ?80 genome

SummaryRegulation of the expression of the tryptophan (trp) operon of Escherichia coli integrated into the genome of transducing phage ϕ80pts was studied by infecting sensitive or immune bacteria and measuring the activities of tryptophan biosynthetic enzymes.In sensitive cells, expression of the trp operon in the genome of a transducing phage ϕ80pt190h is barely repressed by tryptophan and is mostly manifested by the “read through” transcription initiated at the phage promoter site, despite the fact that the phage carries the whole trp operon including the specific operator-promoter region. In ϕ80 lysogenic cells, however, transcription of the “early” genes is repressed by the phage immunity, and the trp operon is completely subject to the control by tryptophan. These results suggest that the repressor of the trp operon cannot prevent its transcription which was initiated at the preceding viral promoter on the same DNA. On the other hand, no appreciable read through transcription occurred with another transducing phage ϕ80pt66 (also carrying the trp operator-promoter region) upon infection of sensitive cells, and the trp operon was under normal control by tryptophan.In relation to these observations, a “turn off” control of early messenger RNA synthesis similar to that found with phage λ was observed in the transcription of the ϕ80 genome.