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M E Armengod

Publications and source records attributed to M E Armengod.

9 recordsLinked to original sources
PubMed1991

Tandem transcription termination sites in the dnaN gene of Escherichia coli.

The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

Base Sequence↗
PubMed1988

Transcriptional organization of the dnaN and recF genes of Escherichia coli K-12.

The dnaN gene of Escherichia coli determines the beta subunit of DNA polymerase III, a multisubunit enzyme responsible for most of the replicative DNA synthesis. The dnaN gene maps between the dnaA and recF genes. We have characterized the regulatory region of the dnaN gene by screening DNA restriction fragments for promoter activity, S1 mapping of mRNAs, deletion analysis, and in vivo dnaN complementation tests. There are at least three dnaN promoters located in the second half of the dnaA coding region. The one closest to the dnaN structural gene is the weakest, but it provides sufficient dnaN expression for complementation when the gene is present on a multicopy plasmid. Deletion of sequences needed for initiation of dnaN translation or introduction of nonsense codons into dnaN causes reduction of recF expression. However, a deletion inactivating dnaN without changing the reading frame of the gene does not affect expression of the recF gene. These results indicate that the dnaN and recF genes are organized in an operon. We have previously shown the presence of termination signals within the dnaN coding region (Armengod, M.E., and Lambíes, E. (1986) Gene (Amst.) 43, 183-196). Therefore, we propose that the polarity produced by nonsense mutations in dnaN is primarily transcriptional. The uncoupling of transcription and translation of the dnaN gene (when translation is interrupted by premature nonsense codons or by other mechanisms) probably results in transcription termination at termination signals in dnaN.

Bacterial Proteins↗
PubMed1986

Overlapping arrangement of the recF and dnaN operons of Escherichia coli; positive and negative control sequences.

The recF gene of Escherichia coli controls one of the recombination pathways and UV sensitivity, but its precise function and expression pattern are still largely unknown. We have characterized the promoter region of the recF gene by mapping for E. coli RNA polymerase binding sites, in vitro transcription experiments, cloning, and S1 mapping of in vivo mRNAs. It contains three overlapping promoters, two initiating transcription towards recF and one in the opposite direction. The recF promoter region is located about 600 bp upstream from the start codon of the recF structural gene and resides entirely within the translated region of the preceding gene, dnaN, which encodes for the beta subunit of DNA polymerase III. This unusual arrangement might provide discoordinate regulation of the recF and dnaN genes, thus controlling the level of DNA polymerase III holoenzyme. Expression of recF is also negatively controlled by sequences located upstream as well as inside the recF coding frame. Such negative regulation may serve to prevent toxic effects due to accumulation of an excessive number of copies of the recF gene product.

Chromosome Mapping↗
PubMed1984

Molecular analysis of the recF gene of Escherichia coli.

We analyzed the nucleotide sequence of a 1.325-kilobase region of wild-type Escherichia coli containing a functional recF gene and six Tn3 mutations that inactivate recF. The analysis shows a potentially translatable reading frame of 1071 nucleotides, which is interrupted by all six insertions. A protein of 40.5 kilodaltons would result from translation of the open reading frame, and a radioactive band of protein of an apparent molecular weight of approximately 40 kilodaltons was seen by the maxicell method using a recF+ plasmid. Putative truncated peptides were seen when two recF::Tn3 mutant plasmids were used. Differential expression of dnaN and recF from a common promoter was noted. recF332::Tn3 was transferred to the chromosome where, in hemizygous condition, it produced UV sensitivity indistinguishable from that produced by two presumed recF point mutations.

Amino Acid Sequence↗
PubMed1981

Role of the recF gene of Escherichia coli K-12 in lambda recombination.

When Escherichia coli K12(lambda) lysogens are infected with heteroimmune lambda phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which lambda replication is blocked, the recombination pathway dependent on the recF gene is fully active in producing viral recombinants even, if the phage is Red+, in the presence of exonuclease I. In contrast, removal of lambda exonuclease and beta protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitor effects of exonuclease I. In the absence of lambda exonuclease, beta protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of beta protein, full efficiency of the RecF pathway can be obtained either via cooperation with lambda exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of lambda exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that lambda exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I. When lambda replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution to the RecF pathway to lambda recombination is observed after removal of the Red system and exonuclease.

Bacteriophage lambda↗
PubMed1978

Influence of the recF143 mutation of Escherichia coli K12 on prophage lambda induction.

Prophage lambda induction in a recF143 mutant of E. coli K12 was studied. The recF143 (lambda) lysogen was inducible by UV irradiation or treatment with mitomycin C. However, the time required for the onset of derepression brought about by these treatments was longer in the recF143 mutant than in rec+ strains, suggesting that the induction pathway was altered in the recF143 mutant. The recF143 (lambda) lysogen was induced at very low doses of UV irradiation or mitomycin C treatment. Moreover, the presence of the recF143 mutation increased the sensitivity to thermal induction of a tif strain.

Coliphages↗
PubMed1976

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage Lambda.

In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage mu Red recombination systems on the rescue of the O plus gene from the prophage by a superinfecting O minus phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants than does the Rec system, and its action requires DNA replication. The presence of UV lesions in the mu DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.

Coliphages↗
PubMedDate not supplied

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Bacterial Proteins↗